Therapeutic CD47 antibodies

ABSTRACT

Provided are monoclonal antibodies and antigen-binding fragments thereof that bind to, and inhibit the activity of, CD47, as well as monoclonal antibodies and antigen binding fragments thereof that compete with the former for binding to CD47. Also provided are combinations of any of the foregoing. Such antibody compounds are variously effective in 1) treating tissue ischemia and ischemia-reperfusion injury (IRI) in the setting of organ preservation and transplantation, pulmonary hypertension, sickle cell disease, myocardial infarction, stroke, and other instances of surgery and/or trauma in which IRI is a component of pathogenesis; 2) in treating autoimmune and inflammatory diseases; and 3) as anti-cancer agents that are toxic to susceptible cancer cells, promoting their phagocytic uptake and clearance, or directly killing such cells.

CROSS-REFERENCE TO A RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.14/940,751, filed Nov. 13, 2015, which is a divisional of U.S. patentapplication Ser. No. 14/104,007, filed Dec. 12, 2013, which claims thebenefit of priority from U.S. Provisional Application No. 61/736,301,filed Dec. 12, 2012, each of which is incorporated herein by referencein its entirety.

SEQUENCE LISTING

The present application is being filed along with a sequence listing inelectronic format. The sequence listing is provided as a file entitled,“VLX0001-201-US 20131212 SequenceListing_ST25”, created on Dec. 11, 2013which is 90 Kb in size. The information in the electronic format of thesequence listing is incorporated herein by reference in its entirety.

The present invention relates to antibodies that bind CD47 and their usein treating conditions and disorders, such as ischemia-reperfusioninjury (IRI) and cancers, mediated by this receptor.

CD47 is a cell surface receptor comprised of an extracellular IgV setdomain, a 5 membrane spanning transmembrane domain, and a cytoplasmictail that is alternatively spliced. Two ligands bind CD47:thrombospondin-1 (TSP1), and signal inhibitory receptor protein alpha(SIRPalpha). TSP1 binding to CD47 activates the heterotrimeric G proteinGi, which leads to suppression of intracellular cyclic AMP (cAMP)levels. In addition, the TSP1-CD47 pathway opposes the beneficialeffects of the nitric oxide pathway in all vascular cells. The nitricoxide (NO) pathway consists of any of three enzymes (nitric oxidesynthases, NOS I, NOS II and NOS III) that generate bioactive gas NOusing arginine as a substrate. NO can act within the cell in which it isproduced, or in neighboring cells, to activate the enzyme solubleguanylyl cyclase that produces the messenger molecule cyclic GMP (cGMP).The proper functioning of the NO-cGMP pathway is essential forprotecting the cardiovascular system against stresses including, but notlimited to, those resulting from wounding, inflammation, hypertension,metabolic syndrome, ischemia, and ischemia-reperfusion injury (IRI). Inthe context of these cellular stresses, the inhibition of the NO-cGMPpathway by the TSP1-CD47 system exacerbates the effects of stress. Thisis a particular problem in the cardiovascular system where both cGMP andcAMP play important protective roles. There are many cases in whichischemia and reperfusion injury cause or contribute to disease, trauma,and poor outcomes of surgical procedures.

SIRPalpha is expressed on hematopoietic cells, including macrophages anddendritic cells. When it engages CD47 on a potential phagocytic targetcell, phagocytosis is slowed or prevented. The CD47-SIRPalphainteraction effectively sends a “don't eat me” signal to the phagocyte.Thus, blocking the SIRPalpha-CD47 interaction with a monoclonal antibodyin this therapeutic context can provide an effective anti-cancer therapyby promoting the uptake and clearance of cancer cells by the host'simmune system. This mechanism is effective in both leukemias and manytypes of solid tumors.

U.S. Pat. No. 8,236,313 contemplates antibodies that could be useful inthe field of ischemia and blood flow to reverse and/or prevent tissueischemia and related and associated tissue and cell damage, includingantibodies that block CD47. No antibodies are actually disclosed.

U.S. Pat. No. 8,101,719 discloses humanized antibodies that bind to CD47for use in treating hematological disorders. Objects of the inventioninclude humanized anti-CD47 antibodies and small antibody fragmentsexhibiting reduced antigenicity while retaining their CD47 bindingactivity and apoptosis-inducing activity. Such antibodies and smallfragments are contemplated for use in treating hematological disorderssuch as various types of leukemias, malignant lymphoma, aplastic anemia,myeodysplastic syndromes, and polycythemia vera. No other properties ofthese antibodies are disclosed.

PCT International Publication WO 2011/143624 discloses chimeric andhumanized anti-CD47 monoclonal antibodies for use as reagents for thediagnosis and immunotherapy of diseases associated with CD47 in humans,particularly in cancer therapy, for example to increase phagocytosis ofcancer cells expressing CD47. Preferred antibodies are non-activating,i.e., block ligand binding, but do not signal. Disclosed humanized B6H12and 5F9 antibodies bound soluble human CD47; B6H12 also bound human CD47on the surface of human CD47-transfected YB2/0 cells. Humanized B6H12and 5F9 antibodies enabled phagocytosis of CFSE-labeled HL-60 cells bymouse bone marrow- or peripheral blood-derived macrophages in vitro,respectively. Humanized B6H12 utilized human VH-3-7 and VK3-11frameworks.

There exists a need for antibodies to human CD47 that selectively blockthe binding of TSP1 to CD47 to promote the beneficial effects of nitricoxide-cGMP signaling and cAMP signaling in the cardiovascular system insettings in which IRI plays a role in pathogenesis. Thesesituations/diseases include organ transplantation, acute kidney injury,cardiopulmonary bypass surgery, pulmonary hypertension, sickle celldisease, myocardial infarction, stroke, surgical resections andreconstructive surgery, reattachment of digits/body parts, skingrafting, and trauma. There is also a need for antibodies that block thebinding of SIRPalpha to CD47, thus providing novel anti-cancertherapies. Such antibodies that also have the ability to selectivelykill transformed or cancer cells are also expected to provide additionaltherapeutic benefit.

Antibody compounds of the present invention meet these needs. They bindto epitopes in the extracellular IgV domain of CD47, inhibiting TSP1 andSIRPalpha binding to CD47 and receptor activation, while inducing littleor no agonist activity. Certain other antibodies of the presentinvention also provide a tumor-toxic effect that is specific toactivated or transformed cells in addition to promoting tumor cellphagocytic clearance. In view of these properties, antibody compounds ofthe present invention should be therapeutically useful in treating manyforms of IRI and both blood cancers and solid tumors.

In addition, the present antibody compounds possess a number of otherdesirable properties, including broad reactivity with CD47 of a widevariety of mammalian species, including that of human, mouse, rat, andpig, making these antibodies useful in both human and veterinarymedicine. This feature is further advantageous in that it facilitatespreclinical studies including, but not limited to, safety and efficacystudies, in a variety of mammalian species, and therefore thedevelopment of such antibodies as human and veterinary therapeutics.

Accordingly, the present invention provides:

EMBODIMENT 1

A monoclonal antibody, or antigen-binding fragment thereof, thatspecifically binds human, rat, mouse, and pig CD47.

EMBODIMENT 2

The monoclonal antibody or antigen-binding fragment thereof ofembodiment 1, which is chimeric or humanized.

EMBODIMENT 3

The monoclonal antibody, or antigen-binding fragment thereof, ofembodiment 1 or embodiment 2, which comprises three light chaincomplementarity determining regions (LCDRs 1-3) and three heavy chaincomplementarity determining regions (HCDRs 1-3), wherein:

LCDR 1 comprises the amino acid sequence RSSQSLVHSNGNTYLH (SEQ ID NO:1)

LCDR 2 comprises the amino acid sequence KVSYRFS (SEQ ID NO:2); and

LCDR 3 comprises the amino acid sequence SQNTHVPRT (SEQ ID NO:3);

HCDR1 comprises the amino acid sequence GYTFTNYYVF (SEQ ID NO:4);

HCDR 2 comprises the amino acid sequence DINPVNGDTNFNEKFKN (SEQ IDNO:5); and

HCDR 3 comprises the amino acid sequence GGYTMDY(SEQ ID NO:6).

EMBODIMENT 4

The monoclonal antibody, or antigen-binding fragment thereof, of any oneof embodiment 1-embodiment 3, which comprises a light chain variableregion (LCVR) and a heavy chain variable region (HCVR), wherein saidLCVR and said HCVR comprise, respectively, amino acid sequences selectedfrom the group consisting of:

-   -   SEQ ID NO:7 and SEQ ID NO:57;    -   SEQ ID NO:8 and SEQ ID NO:58;    -   SEQ ID NO:9 and SEQ ID NO:59;    -   SEQ ID NO:10 and SEQ ID NO:60;    -   SEQ ID NO:11 and SEQ ID NO:61;    -   SEQ ID NO:12 and SEQ ID NO:62;    -   SEQ ID NO:13 and SEQ ID NO:63;    -   SEQ ID NO:14 and SEQ ID NO:64;    -   SEQ ID NO:15 and SEQ ID NO:65;    -   SEQ ID NO:16 and SEQ ID NO:66;    -   SEQ ID NO:17 and SEQ ID NO:67;    -   SEQ ID NO:18 and SEQ ID NO:68;    -   SEQ ID NO:19 and SEQ ID NO:69;    -   SEQ ID NO:20 and SEQ ID NO:70;    -   SEQ ID NO:21 and SEQ ID NO:71;    -   SEQ ID NO:22 and SEQ ID NO:72;    -   SEQ ID NO:23 and SEQ ID NO:73;    -   SEQ ID NO:24 and SEQ ID NO:74;    -   SEQ ID NO:25 and SEQ ID NO:75;    -   SEQ ID NO:26 and SEQ ID NO:76;    -   SEQ ID NO:27 and SEQ ID NO:77;    -   SEQ ID NO:28 and SEQ ID NO:78;    -   SEQ ID NO:29 and SEQ ID NO:79;    -   SEQ ID NO:30 and SEQ ID NO:80; and    -   SEQ ID NO:31 and SEQ ID NO:81.

EMBODIMENT 5

A monoclonal antibody, or antigen-binding fragment thereof, whichcomprises a light chain variable region (LCVR) and a heavy chainvariable region (HCVR), wherein said LCVR and said HCVR comprise,respectively, amino acid sequences selected from the group consistingof:

-   -   SEQ ID NO:7 and SEQ ID NO:57;    -   SEQ ID NO:8 and SEQ ID NO:58;    -   SEQ ID NO:11 and SEQ ID NO:61;    -   SEQ ID NO:14 and SEQ ID NO:64;    -   SEQ ID NO:16 and SEQ ID NO:66;    -   SEQ ID NO:18 and SEQ ID NO:68;    -   SEQ ID NO:19 and SEQ ID NO:69;    -   SEQ ID NO:25 and SEQ ID NO:75;    -   SEQ ID NO:27 and SEQ ID NO:77;    -   SEQ ID NO:28 and SEQ ID NO:78;    -   SEQ ID NO:29 and SEQ ID NO:79;    -   SEQ ID NO:30 and SEQ ID NO:80; and    -   SEQ ID NO:31 and SEQ ID NO:81.

EMBODIMENT 6

A monoclonal antibody, or antigen-binding fragment thereof, whichcomprises a light chain variable region (LCVR) and a heavy chainvariable region (HCVR), wherein said LCVR and said HCVR comprise,respectively, amino acid sequences selected from the group consistingof:

-   -   SEQ ID NO:9 and SEQ ID NO:59;    -   SEQ ID NO:10 and SEQ ID NO:60;    -   SEQ ID NO:12 and SEQ ID NO:62;    -   SEQ ID NO:13 and SEQ ID NO:63;    -   SEQ ID NO:15 and SEQ ID NO:65;    -   SEQ ID NO:17 and SEQ ID NO:67;    -   SEQ ID NO:20 and SEQ ID NO:70;    -   SEQ ID NO:21 and SEQ ID NO:71;    -   SEQ ID NO:22 and SEQ ID NO:72;    -   SEQ ID NO:23 and SEQ ID NO:73;    -   SEQ ID NO:24 and SEQ ID NO:74; and    -   SEQ ID NO:26 and SEQ ID NO:76.

EMBODIMENT 7

A monoclonal antibody, or antigen-binding fragment thereof, thatcompetes with said monoclonal antibody or antigen-binding fragmentthereof of any one of embodiment 1-embodiment 6 for binding to CD47.

EMBODIMENT 8

A pharmaceutical composition, comprising said monoclonal antibody, orantigen-binding fragment thereof, of any one of embodiment 1-embodiment7, and a pharmaceutically or physiologically acceptable carrier,diluent, or excipient.

EMBODIMENT 9

A monoclonal antibody, or antigen-binding fragment thereof, of any oneof embodiment 1-embodiment 7 for use in human therapy or therapy ofcompanion/pet animals, working animals, sport animals, zoo animals, ortherapy of other valuable animals kept in captivity.

EMBODIMENT 10

The monoclonal antibody, or antigen-binding fragment thereof, of any oneof embodiment 1-embodiment 7 for use in treating ischemia-reperfusioninjury, or an autoimmune or inflammatory disease, in a human orcompanion/pet animal, working animal, sport animal, zoo animal, or othervaluable animal kept in captivity.

EMBODIMENT 11

The monoclonal antibody, or antigen-binding fragment thereof, ofembodiment 10, which comprises a light chain variable region (LCVR) anda heavy chain variable region (HCVR), wherein said LCVR and said HCVRcomprise, respectively, amino acid sequences selected from the groupconsisting of:

-   -   SEQ ID NO:7 and SEQ ID NO:57;    -   SEQ ID NO:8 and SEQ ID NO:58;    -   SEQ ID NO:11 and SEQ ID NO:61;    -   SEQ ID NO:14 and SEQ ID NO:64;    -   SEQ ID NO:16 and SEQ ID NO:66;    -   SEQ ID NO:18 and SEQ ID NO:68;    -   SEQ ID NO:19 and SEQ ID NO:69;    -   SEQ ID NO:25 and SEQ ID NO:75;    -   SEQ ID NO:27 and SEQ ID NO:77;    -   SEQ ID NO:28 and SEQ ID NO:78;    -   SEQ ID NO:29 and SEQ ID NO:79;    -   SEQ ID NO:30 and SEQ ID NO:80; and    -   SEQ ID NO:31 and SEQ ID NO:81.

EMBODIMENT 12

The monoclonal antibody, or antigen-binding fragment thereof, ofembodiment 10 or embodiment 11, wherein said ischemia-reperfusion injuryoccurs in organ transplantation, acute kidney injury, cardiopulmonarybypass surgery, pulmonary hypertension, sickle cell disease, myocardialinfarction, stroke, surgical resections and reconstructive surgery,reattachment of an appendage or other body part, skin grafting, ortrauma.

EMBODIMENT 13

The monoclonal antibody, or antigen-binding fragment thereof, ofembodiment 10 or 11, wherein said autoimmune or inflammatory disease isselected from the group consisting of arthritis, multiple sclerosis,psoriasis, Crohn's disease, inflammatory bowel disease, lupus, Grave'sdisease and Hashimoto's thyroiditis, and ankylosing spondylitis.

EMBODIMENT 14

The monoclonal antibody, or antigen-binding fragment thereof, of any oneof embodiment 1-embodiment 7 for use in treating a susceptible cancer.

EMBODIMENT 15

The monoclonal antibody, or antigen-binding fragment thereof, ofembodiment 14, which comprises a light chain variable region (LCVR) anda heavy chain variable region (HCVR), wherein said LCVR and said HCVRcomprise, respectively, amino acid sequences selected from the groupconsisting of:

-   -   SEQ ID NO:9 and SEQ ID NO:59;    -   SEQ ID NO:10 and SEQ ID NO:60;    -   SEQ ID NO:12 and SEQ ID NO:62;    -   SEQ ID NO:13 and SEQ ID NO:63;    -   SEQ ID NO:15 and SEQ ID NO:65;    -   SEQ ID NO:17 and SEQ ID NO:67;    -   SEQ ID NO:20 and SEQ ID NO:70;    -   SEQ ID NO:21 and SEQ ID NO:71;    -   SEQ ID NO:22 and SEQ ID NO:72;    -   SEQ ID NO:23 and SEQ ID NO:73;    -   SEQ ID NO:24 and SEQ ID NO:74; and    -   SEQ ID NO:26 and SEQ ID NO:76.

EMBODIMENT 16

The monoclonal antibody, or antigen binding fragment thereof, ofembodiment 14 or embodiment 15, which promotes phagocytosis and/orkilling of cells of said susceptible cancer.

EMBODIMENT 17

The monoclonal antibody, or antigen binding fragment thereof, of any oneof embodiment 14-embodiment 16, wherein said susceptible cancer isselected from the group consisting of a leukemia, a lymphoma, ovariancancer, breast cancer, endometrial cancer, colon cancer, rectal cancer,bladder cancer, lung cancer, bronchial cancer, bone cancer, prostatecancer, pancreatic cancer, liver and bile duct cancer, esophagealcancer, renal cancer, thyroid cancer, head and neck cancer, testicularcancer, glioblastoma, astrocytoma, melanoma, and leiomyosarcoma.

EMBODIMENT 18

The monoclonal antibody, or antigen binding fragment thereof, ofembodiment 17, wherein said leukemia is selected from the groupconsisting of acute lymphocytic (lymphoblastic) leukemia, acute myeloidleukemia, chronic lymphocytic leukemia, multiple myeloma, and chronicmyeloid leukemia.

EMBODIMENT 19

Use of said monoclonal antibody, or antigen-binding fragment thereof, ofany one of embodiment 1-embodiment 7 to treat ischemia-reperfusioninjury, or an autoimmune or inflammatory disease, in a human orcompanion/pet animal, working animal, sport animal, zoo animal, or othervaluable animal kept in captivity.

EMBODIMENT 20

The use of embodiment 19, wherein said monoclonal antibody, or antigenbinding fragment thereof, comprises a light chain variable region (LCVR)and a heavy chain variable region (HCVR), wherein said LCVR and saidHCVR comprise, respectively, amino acid sequences selected from thegroup consisting of:

-   -   SEQ ID NO:7 and SEQ ID NO:57;    -   SEQ ID NO:8 and SEQ ID NO:58;    -   SEQ ID NO:11 and SEQ ID NO:61;    -   SEQ ID NO:14 and SEQ ID NO:64;    -   SEQ ID NO:16 and SEQ ID NO:66;    -   SEQ ID NO:18 and SEQ ID NO:68;    -   SEQ ID NO:19 and SEQ ID NO:69;    -   SEQ ID NO:25 and SEQ ID NO:75;    -   SEQ ID NO:27 and SEQ ID NO:77;    -   SEQ ID NO:28 and SEQ ID NO:78;    -   SEQ ID NO:29 and SEQ ID NO:79;    -   SEQ ID NO:30 and SEQ ID NO:80; and    -   SEQ ID NO:31 and SEQ ID NO:81.

EMBODIMENT 21

Use of said monoclonal antibody, or antigen-binding fragment thereof, ofany one of embodiment 1-embodiment 7 to treat a susceptible cancer.

EMBODIMENT 22

The use of embodiment 21, wherein said monoclonal antibody, or antigenbinding fragment thereof, comprises a light chain variable region (LCVR)and a heavy chain variable region (HCVR), wherein said LCVR and saidHCVR comprise, respectively, amino acid sequences selected from thegroup consisting of:

-   -   SEQ ID NO:9 and SEQ ID NO:59;    -   SEQ ID NO:10 and SEQ ID NO:60;    -   SEQ ID NO:12 and SEQ ID NO:62;    -   SEQ ID NO:13 and SEQ ID NO:63;    -   SEQ ID NO:15 and SEQ ID NO:65;    -   SEQ ID NO:17 and SEQ ID NO:67;    -   SEQ ID NO:20 and SEQ ID NO:70;    -   SEQ ID NO:21 and SEQ ID NO:71;    -   SEQ ID NO:22 and SEQ ID NO:72;    -   SEQ ID NO:23 and SEQ ID NO:73;    -   SEQ ID NO:24 and SEQ ID NO:74; and    -   SEQ ID NO:26 and SEQ ID NO:76.

EMBODIMENT 23

Use of said monoclonal antibody, or antigen-binding fragment thereof, ofany one of embodiment 1-embodiment 7 for the manufacture of a medicamentto treat ischemia-reperfusion injury, or an autoimmune or inflammatorydisease, in a human or companion/pet animal, working animal, sportanimal, zoo animal, or other valuable animal kept in captivity.

EMBODIMENT 24

The use of embodiment 23, wherein said monoclonal antibody, or antigenbinding fragment thereof, comprises a light chain variable region (LCVR)and a heavy chain variable region (HCVR), wherein said LCVR and saidHCVR comprise, respectively, amino acid sequences selected from thegroup consisting of:

-   -   SEQ ID NO:7 and SEQ ID NO:57;    -   SEQ ID NO:8 and SEQ ID NO:58;    -   SEQ ID NO:11 and SEQ ID NO:61;    -   SEQ ID NO:14 and SEQ ID NO:64;    -   SEQ ID NO:16 and SEQ ID NO:66;    -   SEQ ID NO:18 and SEQ ID NO:68;    -   SEQ ID NO:19 and SEQ ID NO:69;    -   SEQ ID NO:25 and SEQ ID NO:75;    -   SEQ ID NO:27 and SEQ ID NO:77;    -   SEQ ID NO:28 and SEQ ID NO:78;    -   SEQ ID NO:29 and SEQ ID NO:79;    -   SEQ ID NO:30 and SEQ ID NO:80; and    -   SEQ ID NO:31 and SEQ ID NO:81.

EMBODIMENT 25

The use of embodiment 23 or embodiment 24, wherein saidischemia-reperfusion injury occurs in organ transplantation, acutekidney injury, cardiopulmonary bypass surgery, pulmonary hypertension,sickle cell disease, myocardial infarction, stroke, surgical resectionsand reconstructive surgery, reattachment of an appendage or other bodypart, skin grafting, and trauma.

EMBODIMENT 26

The use of embodiment 23 or embodiment 24, wherein said autoimmune orinflammatory disease is selected from the group consisting of arthritis,multiple sclerosis, psoriasis, Crohn's disease, inflammatory boweldisease, lupus, Grave's disease and Hashimoto's thyroiditis, andankylosing spondylitis.

EMBODIMENT 27

Use of said monoclonal antibody, or antigen-binding fragment thereof, ofany one of embodiment 1-embodiment 7 for the manufacture of a medicamentto treat a susceptible cancer.

EMBODIMENT 28

The use of embodiment 27, wherein said monoclonal antibody, orantigen-binding fragment thereof, comprises a light chain variableregion (LCVR) and a heavy chain variable region (HCVR), wherein saidLCVR and said HCVR comprise, respectively, amino acid sequences selectedfrom the group consisting of:

-   -   SEQ ID NO:9 and SEQ ID NO:59;    -   SEQ ID NO:10 and SEQ ID NO:60;    -   SEQ ID NO:12 and SEQ ID NO:62;    -   SEQ ID NO:13 and SEQ ID NO:63;    -   SEQ ID NO:15 and SEQ ID NO:65;    -   SEQ ID NO:17 and SEQ ID NO:67;    -   SEQ ID NO:20 and SEQ ID NO:70;    -   SEQ ID NO:21 and SEQ ID NO:71;    -   SEQ ID NO:22 and SEQ ID NO:72;    -   SEQ ID NO:23 and SEQ ID NO:73;    -   SEQ ID NO:24 and SEQ ID NO:74; and    -   SEQ ID NO:26 and SEQ ID NO:76.

EMBODIMENT 29

A method of treating ischemia or ischemia-reperfusion injury in apatient in need thereof, comprising administering to said patient aneffective amount of said monoclonal antibody, or antigen-bindingfragment thereof, of any one of embodiment 1-embodiment 7, or amonoclonal antibody, or antigen-binding fragment thereof, that competeswith said monoclonal antibody or antigen-binding fragment thereof of anyone of embodiment 1-embodiment 7 for binding to CD47.

EMBODIMENT 30

The method of embodiment 29, wherein said patient is about to besubjected to, or is experiencing, ischemia or ischemia-reperfusioninjury.

EMBODIMENT 31

The method of embodiment 29 or embodiment 30, wherein said patient is ahuman.

EMBODIMENT 32

The method of embodiment 29 or embodiment 30, wherein said patient is acompanion/pet animal, working animal, sport animal, zoo animal, or othervaluable animal kept in captivity.

EMBODIMENT 33

The method of any one of embodiment 29-embodiment 32, wherein saidischemia occurs because said patient will undergo, or is undergoing, asurgery selected from the group consisting of integument surgery, softtissue surgery, composite tissue surgery, cosmetic surgery, surgicalresections, reconstructive surgery, skin graft surgery, and limbreattachment surgery.

EMBODIMENT 34

The method of embodiment 33, wherein said skin graft is an autograft.

EMBODIMENT 35

The method of any one of embodiment 29-32, wherein said ischemia occursbecause said patient will undergo, or is undergoing, organ transplantsurgery.

EMBODIMENT 36

The method of any one of embodiment 29-embodiment 32, wherein saidischemia-reperfusion injury occurs in organ transplantation, acutekidney injury, cardiopulmonary bypass surgery, pulmonary hypertension,sickle cell disease, myocardial infarction, stroke, surgical resection,reconstructive surgery, reattachment of an appendage or other body part,or skin grafting.

EMBODIMENT 37

The method of any one of embodiment 29-embodiment 36, wherein saidmonoclonal antibody, antigen-binding fragment thereof, or competingmonoclonal antibody or antigen binding fragment thereof, is administeredbefore, during, or after said subject undergoes ischemia or surgery, ora combination of any of these time periods.

EMBODIMENT 38

The method of any one of embodiment 29-embodiment 37, further comprisingadministering to said patient an effective amount of a nitric oxidedonor, precursor, or both.

EMBODIMENT 39

The method of embodiment 38, wherein said nitric oxide donor orprecursor is selected from the group consisting of NO gas, isosorbidedinitrite, nitrite, nitroprusside, nitroglycerin,3-Morpholinosydnonimine (SIN-1), S-nitroso-N-acetylpenicillamine (SNAP),Diethylenetriamine/NO (DETA/NO), S-nitrosothiols, and arginine.

EMBODIMENT 40

A method of increasing tissue perfusion in a subject in need thereof,comprising administering to said subject an effective amount of amonoclonal antibody, or antigen-binding fragment thereof, of any one ofembodiment 1-embodiment 7, or a monoclonal antibody, or antigen-bindingfragment thereof, that competes with said monoclonal antibody orantigen-binding fragment thereof of any one of embodiment 1-embodiment 7for binding to CD47.

EMBODIMENT 41

The method of embodiment 40, wherein said subject has, or is at risk ofdeveloping, at least one disease or condition selected from the groupconsisting of ischemia-reperfusion injury, myocardial infarction,myocardial ischemia, stroke, cerebral ischemia, sickle cell anemia, andpulmonary hypertension.

EMBODIMENT 42

The method of embodiment 40, wherein said subject has, or is at risk ofdeveloping, at least one disease or condition selected from the groupconsisting of hypertension, atherosclerosis, vasculopathy, ischemiasecondary to diabetes, and peripheral vascular disease.

EMBODIMENT 43

The method of embodiment 40, wherein the need for increased tissueperfusion arises because said subject has had, is having, or will have,a surgery selected from the group consisting of integument surgery, softtissue surgery, composite tissue surgery, skin graft surgery, resectionof a solid organ, and reattachment or an appendage or other body part.

EMBODIMENT 44

The method of embodiment 43, wherein said skin graft is an autograft.

EMBODIMENT 45

The method of embodiment 40, wherein the need for increased tissueperfusion arises because said subject has had, is having, or will have,organ transplant surgery.

EMBODIMENT 46

The method of any one of embodiment 40-embodiment 45, further comprisingadministering to said subject an effective amount of a nitric oxidedonor, precursor, or both.

EMBODIMENT 47

The method of embodiment 46, wherein said nitric oxide donor orprecursor is selected from the group consisting of NO gas, isosorbidedinitrite, nitrite, nitroprusside, nitroglycerin,3-Morpholinosydnonimine (SIN-1), S-nitroso-N-acetylpenicillamine (SNAP),Diethylenetriamine/NO (DETA/NO), S-nitrosothiols, and arginine.

EMBODIMENT 48

A method of transplanting a donor organ from an organ donor to an organrecipient, comprising any single step, any combination of steps, or allsteps selected from the group consisting of steps i)-iii):

i) administering to said organ donor prior to, during, both prior to andduring, after, or any combination thereof, donation of said donor organan effective amount of said monoclonal antibody or antigen-bindingfragment thereof of any one of embodiment 1-embodiment 7, and/or amonoclonal antibody, or antigen-binding fragment thereof, that competeswith said monoclonal antibody or antigen-binding fragment thereof of anyone of embodiment 1-embodiment 7 for binding to CD47;

ii) contacting said donor organ prior to, during, both prior to andduring, after, or any combination thereof, transplantation to said organrecipient, and an effective amount of said monoclonal antibody orantigen-binding fragment thereof of any one of embodiment 1-embodiment7, and/or a monoclonal antibody, or antigen-binding fragment thereof,that competes with said monoclonal antibody or antigen-binding fragmentthereof of any one of embodiment 1-embodiment 7 for binding to CD47; and

iii) administering to said organ recipient prior to, during, both priorto and during, after, or any combination thereof, transplantation ofsaid donor organ to said organ recipient, an effective amount of saidmonoclonal antibody or antigen-binding fragment thereof of any one of[1]-[7], and/or a monoclonal antibody, or antigen-binding fragmentthereof, that competes with said monoclonal antibody or antigen-bindingfragment thereof of any one of embodiment 1-embodiment 7 for binding toCD47.

EMBODIMENT 49

The method of embodiment 48, wherein said monoclonal antibody orantigen-binding fragment thereof of any one of embodiment 1-embodiment7, or monoclonal antibody, or antigen-binding fragment thereof, thatcompetes with said monoclonal antibody or antigen-binding fragmentthereof of any one of embodiment 1-embodiment 7 for binding to CD47,reduces ischemia reperfusion injury in said donor organ.

EMBODIMENT 50

The method of embodiment 48 or embodiment 49, further comprisingadministering to said organ donor, said donor organ, said organrecipient, or any combination thereof, an effective amount of a nitricoxide donor, precursor, or both.

EMBODIMENT 51

The method of embodiment 50, wherein said nitric oxide donor orprecursor is selected from the group consisting of NO gas, isosorbidedinitrite, nitrite, nitroprusside, nitroglycerin,3-Morpholinosydnonimine (SIN-1), S-nitroso-N-acetylpenicillamine (SNAP),Diethylenetriamine/NO (DETA/NO), S-nitrosothiols, and arginine.

EMBODIMENT 52

A method of treating an autoimmune or inflammatory disease in a patientin need thereof, comprising administering to said patient an effectiveamount of said monoclonal antibody, or antigen-binding fragment thereof,of any one of embodiment 1-embodiment 7, or a monoclonal antibody, orantigen-binding fragment thereof, that competes with said monoclonalantibody or antigen-binding fragment thereof of any one of [1]-[7] forbinding to CD47.

EMBODIMENT 53

The method of embodiment 52, wherein said autoimmune or inflammatorydisease is selected from the group consisting of arthritis, multiplesclerosis, psoriasis, Crohn's disease, inflammatory bowel disease,lupus, Grave's disease and Hashimoto's thyroiditis, and ankylosingspondylitis.

EMBODIMENT 54

The method of embodiment 52 or embodiment 53, wherein said patient is ahuman.

EMBODIMENT 55

The method of embodiment 52 or embodiment 53, wherein said patient is acompanion/pet animal, working animal, sport animal, zoo animal, or othervaluable animal kept in captivity.

EMBODIMENT 56

The method of any one of embodiment 52-embodiment 55, further comprisingadministering to said patient an effective amount of a nitric oxidedonor, precursor, or both.

EMBODIMENT 57

The method of embodiment 56, wherein said nitric oxide donor orprecursor is selected from the group consisting of NO gas, isosorbidedinitrite, nitrite, nitroprusside, nitroglycerin,3-Morpholinosydnonimine (SIN-1), S-nitroso-N-acetylpenicillamine (SNAP),Diethylenetriamine/NO (DETA/NO), S-nitrosothiols, and arginine.

EMBODIMENT 58

A method of treating a susceptible cancer in a human or companion/petanimal, working animal, sport animal, zoo animal, or other valuableanimal kept in captivity in need thereof, comprising administeringthereto an effective amount of a monoclonal antibody or antigen bindingfragment thereof of any one of embodiment 1-embodiment 7, or amonoclonal antibody, or antigen-binding fragment thereof, that competeswith said monoclonal antibody or antigen-binding fragment thereof of anyone of embodiment 1-embodiment 7 for binding to CD47, and which exhibitscytotoxic activity.

EMBODIMENT 59

The method of embodiment 58, wherein said susceptible cancer is selectedfrom the group consisting of a leukemia, a lymphoma, ovarian cancer,breast cancer, endometrial cancer, colon cancer, rectal cancer, bladdercancer, lung cancer, bronchial cancer, bone cancer, prostate cancer,pancreatic cancer, liver and bile duct cancer, esophageal cancer, renalcancer, thyroid cancer, head and neck cancer, testicular cancer,glioblastoma, astrocytoma, melanoma, and leiomyosarcoma.

EMBODIMENT 60

The method of embodiment 59, wherein said leukemia is selected from thegroup consisting of acute lymphocytic (lymphoblastic) leukemia, acutemyeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, andchronic myeloid leukemia.

EMBODIMENT 61

The method of any one of embodiment 58-embodiment 60, wherein saidmonoclonal antibody or antigen-binding fragment thereof of any one ofembodiment 1-embodiment 7, or said monoclonal antibody, orantigen-binding fragment thereof, that competes with said monoclonalantibody or antigen-binding fragment thereof of any one of embodiment1-embodiment 7 for binding to CD47 and which exhibits cytotoxicactivity, increases phagocytosis of cells of said susceptible cancer.

EMBODIMENT 62

The method of embodiment 61, wherein said monoclonal antibody orantigen-binding fragment thereof of any one of embodiment 1-embodiment7, or said monoclonal antibody, or antigen-binding fragment thereof,that competes with said monoclonal antibody or antigen-binding fragmentthereof of any one of embodiment 1-embodiment 7 for binding to CD47which exhibits cytotoxic activity and increases phagocytosis of cells ofsaid susceptible cancer inhibits CD47 binding to SIRPalpha.

EMBODIMENT 63

The method of any one of embodiment 58-embodiment 62, wherein saidmonoclonal antibody or antigen-binding fragment thereof of any one ofembodiment 1-embodiment 7, or said monoclonal antibody, orantigen-binding fragment thereof, that competes with said monoclonalantibody or antigen-binding fragment thereof of any one of embodiment1-embodiment 7 for binding to CD47 and which exhibits cytotoxicactivity, is directly toxic to cells of said susceptible cancer.

Further scope of the applicability of the present invention will becomeapparent from the detailed description provided below. However, itshould be understood that the detailed description and specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

The following detailed description of the invention is provided to aidthose skilled in the art in practicing the present invention. Even so,the following detailed description should not be construed to undulylimit the present invention, as modifications and variations in theembodiments herein discussed may be made by those of ordinary skill inthe art without departing from the spirit or scope of the presentinventive discovery.

Definitions

A full-length antibody as it exists naturally is an immunoglobulinmolecule comprising two heavy (H) chains and two light (L) chainsinterconnected by disulfide bonds. The amino terminal portion of eachchain includes a variable region of about 100-110 or more amino acidsprimarily responsible for antigen recognition via the complementaritydetermining regions (CDRs) contained therein. The carboxy-terminalportion of each chain defines a constant region primarily responsiblefor effector function.

The CDRs are interspersed with regions that are more conserved, termedframework regions (“FR”). Each light chain variable region (LCVR) andheavy chain variable region (HCVR) is composed of 3 CDRs and 4 FRs,arranged from amino-terminus to carboxy-terminus in the following order:FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The 3 CDRs of the light chain arereferred to as “LCDR1, LCDR2, and LCDR3” and the 3 CDRs of the heavychain are referred to as “HCDR1, HCDR2, and HCDR3.” The CDRs containmost of the residues which form specific interactions with the antigen.The numbering and positioning of CDR amino acid residues within the LCVRand HCVR regions are in accordance with the well-known Kabat numberingconvention.

Light chains are classified as kappa or lambda, and are characterized bya particular constant region as known in the art. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, and define theisotope of an antibody as IgG, IgM, IgA, IgD, or IgE, respectively. IgGantibodies can be further divided into subclasses, e.g., IgG1, IgG2,IgG3, IgG4. Each heavy chain type is characterized by a particularconstant region with a sequence well known in the art.

As used herein, the term “monoclonal antibody” (mAb) as applied to thepresent antibody compounds refers to an antibody that is derived from asingle copy or clone including, for example, any eukaryotic,prokaryotic, or phage clone, and not the method by which it is produced.mAbs of the present invention preferably exist in a homogeneous orsubstantially homogeneous population. Complete mAbs contain two heavychains and two light chains. “Antigen binding fragments” of suchmonoclonal antibodies include, for example, Fab fragments, Fab′fragments, F(ab′)₂ fragments, single chain Fv fragments, and one-armedantibodies comprising a light chain and a heavy chain. Monoclonalantibodies and antigen-binding fragments thereof of the presentinvention can be produced, for example, by recombinant technologies,phage display technologies, synthetic technologies, e.g., CDR-grafting,or combinations of such technologies, or other technologies known in theart.

“Antibody compounds” refers to mAbs and Fabs, and competing antibodies,disclosed herein. Additional antibody compounds exhibiting similarfunctional properties according to the present invention can begenerated by conventional methods. For example, mice can be immunizedwith human CD47 or fragments thereof, the resulting antibodies can berecovered and purified, and determination of whether they possessbinding and functional properties similar to or the same as the antibodycompounds disclosed herein can be assessed by the methods disclosed inExamples 3 and 4, below. Antigen-binding fragments can also be preparedby conventional methods. Methods for producing and purifying antibodiesand antigen-binding fragments are well known in the art and can befound, for example, in Harlow and Lane (1988) Antibodies, A LaboratoryManual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,chapters 5-8 and 15, ISBN 0-87969-314-2.

The phrase “humanized antibodies” refers to monoclonal antibodies andantigen binding fragments thereof in addition to the antibody compoundsdisclosed herein that have binding and functional properties accordingto the invention similar to those disclosed herein, and that haveframework regions that are substantially human or fully humansurrounding CDRs derived from a non-human antibody. “Framework region”or “framework sequence” refers to any one of framework regions 1 to 4.Humanized antibodies and antigen binding fragments encompassed by thepresent invention include molecules wherein any one or more of frameworkregions 1 to 4 is substantially or fully human, i.e., wherein any of thepossible combinations of individual substantially or fully humanframework regions 1 to 4, is present. For example, this includesmolecules in which framework region 1 and framework region 2, frameworkregion 1 and framework region 3, framework region 1, 2, and 3, etc., aresubstantially or fully human. Substantially human frameworks are thosethat have at least about 80% sequence identity to a known human germlineframework sequence. Preferably, the substantially human frameworks haveat least about 85%, at least about 90%, at least about 95%, or at leastabout 99% sequence identity to a known human germline frameworksequence.

Fully human frameworks are those that are identical to a known humangermline framework sequence. Human framework germline sequences can beobtained from ImMunoGeneTics (IMGT) via their website or from TheImmunoglobulin FactsBook by Marie-Paule Lefranc and Gerard Lefranc,Academic Press, 2001, ISBN 012441351. For example, germline light chainframeworks can be selected from the group consisting of: A11, A17, A18,A19, A20, A27, A30, LI, L1I, L12, L2, L5, L15, L6, L8, O12, O2, and O8,and germline heavy chain framework regions can be selected from thegroup consisting of: VH2-5, VH2-26, VH2-70, VH3-20, VH3-72, VHI-46,VH3-9, VH3-66, VH3-74, VH4-31, VHI-18, VHI-69, VI-13-7, VH3-11, VH3-15,VH3-21, VH3-23, VH3-30, VH3-48, VH4-39, VH4-59, and VH5-5I.

Humanized antibodies in addition to those disclosed herein exhibitingsimilar functional properties according to the present invention can begenerated using several different methods. In one approach, the parentantibody compound CDRs are grafted into a human framework that has ahigh sequence identity with the parent antibody compound framework. Thesequence identity of the new framework will generally be at least about80%, at least about 85%, at least about 90%, at least about 95%, or atleast about 99% identical to the sequence of the corresponding frameworkin the parent antibody compound. In the case of frameworks having fewerthan 100 amino acid residues, one, two, or three amino acid residues canbe changed. This grafting may result in a reduction in binding affinitycompared to that of the parent antibody. If this is the case, theframework can be back-mutated to the parent framework at certainpositions based on specific criteria disclosed by Queen et al. (1991)Proc. Natl. Acad. Sci. USA 88:2869. Additional references describingmethods useful in humanizing mouse antibodies include U.S. Pat. Nos.4,816,397; 5,225,539; and 5,693,761; computer programs ABMOD and ENCADas described in Levitt (1983) J. Mol. Biol. 168:595-620; and the methodof Winter and co-workers (Jones et al. (1986) Nature 321:522-525;Riechmann et al. (1988) Nature 332:323-327; and Verhoeyen et al. (1988)Science 239:1534-1536.

The identification of residues to consider for back-mutation can becarried out as follows.

When an amino acid falls under the following category, the frameworkamino acid of the human germ-line sequence that is being used (the“acceptor framework”) is replaced by a framework amino acid from aframework of the parent antibody compound (the “donor framework”): (a)the amino acid in the human framework region of the acceptor frameworkis unusual for human frameworks at that position, whereas thecorresponding amino acid in the donor immunoglobulin is typical forhuman frameworks at that position; (b) the position of the amino acid isimmediately adjacent to one of the CDRs; or (c) any side chain atom of aframework amino acid is within about 5-6 angstroms (center-to-center) ofany atom of a CDR amino acid in a three dimensional immunoglobulinmodel.

When each of the amino acids in the human framework region of theacceptor framework and a corresponding amino acid in the donor frameworkis generally unusual for human frameworks at that position, such aminoacid can be replaced by an amino acid typical for human frameworks atthat position. This back-mutation criterion enables one to recover theactivity of the parent antibody compound.

Another approach to generating human engineered antibodies exhibitingsimilar functional properties to the antibody compounds disclosed hereininvolves randomly mutating amino acids within the grafted CDRs withoutchanging the framework, and screening the resultant molecules forbinding affinity and other functional properties that are as good as orbetter than those of the parent antibody compounds. Single mutations canalso be introduced at each amino acid position within each CDR, followedby assessing the effects of such mutations on binding affinity and otherfunctional properties. Single mutations producing improved propertiescan be combined to assess their effects in combination with one another.

Further, a combination of both of the foregoing approaches is possible.After CDR grafting, one can back-mutate specific framework regions inaddition to introducing amino acid changes in the CDRs. This methodologyis described in Wu et al. (1999) J. Mol. Biol. 294:151-162.

The method described in Example 1 below can also be employed.

Applying the teachings of the present invention, a person skilled in theart can use common techniques, e.g., site-directed mutagenesis, tosubstitute amino acids within the presently disclosed CDR and frameworksequences and thereby generate further variable region amino acidsequences derived from the present sequences. Up to all naturallyoccurring amino acids can be introduced at a specific substitution site.The methods disclosed herein can then be used to screen these additionalvariable region amino acid sequences to identify sequences having theindicated in vivo functions. In this way, further sequences suitable forpreparing human engineered antibodies and antigen-binding portionsthereof in accordance with the present invention can be identified.Preferably, amino acid substitution within the frameworks is restrictedto one, two, or three positions within any one or more of the 4 lightchain and/or heavy chain framework regions disclosed herein. Preferably,amino acid substitution within the CDRs is restricted to one, two, orthree positions within any one or more of the 3 light chain and/or heavychain CDRs. Combinations of the various changes within these frameworkregions and CDRs described above are also possible.

That the functional properties of the antibody compounds generated byintroducing the amino acid modifications discussed above conform tothose exhibited by the specific molecules disclosed herein can beconfirmed by the methods disclosed below in Examples 3 and 4.

The terms “specifically binds”, “bind specifically”, “specific binding”,and the like as applied to the present antibody compounds refer to theability of a specific binding agent (such as an antibody) to bind to atarget molecular species in preference to binding to other molecularspecies with which the specific binding agent and target molecularspecies are admixed. A specific binding agent is said specifically torecognize a target molecular species when it can bind specifically tothat target.

“Binding affinity” is a term that refers to the strength of binding ofone molecule to another at a site on the molecule. If a particularmolecule will bind to or specifically associate with another particularmolecule, these two molecules are said to exhibit binding affinity foreach other. Binding affinity is related to the association constant anddissociation constant for a pair of molecules, but it is not critical tothe methods herein that these constants be measured or determined.Rather, affinities as used herein to describe interactions betweenmolecules of the described methods are generally apparent affinities(unless otherwise specified) observed in empirical studies, which can beused to compare the relative strength with which one molecule (e.g., anantibody or other specific binding partner) will bind two othermolecules (e.g., two versions or variants of a peptide). The concepts ofbinding affinity, association constant, and dissociation constant arewell known.

The term “epitope” refers to a specific arrangement of amino acidslocated on a peptide or protein to which an antibody or antibodyfragment binds. Epitopes often consist of a chemically active surfacegrouping of molecules such as amino acids or sugar side chains, and havespecific three dimensional structural characteristics as well asspecific charge characteristics. Epitopes can be linear, i.e., involvingbinding to a single sequence of amino acids, or conformational, i.e.,involving binding to two or more sequences of amino acids in variousregions of the antigen that may not necessarily be contiguous.

Monoclonal antibodies or antigen-binding fragments thereof that“compete” with the molecules disclosed herein are those that bind humanCD47 at site(s) that are identical to, or overlapping with, the site(s)at which the present molecules bind. Competing monoclonal antibodies orantigen-binding fragments thereof can be identified, for example, via anantibody competition assay. For example, a sample of purified orpartially purified human CD47 extracellular domain can be bound to asolid support. Then, an antibody compound, or antigen binding fragmentthereof, of the present invention and a monoclonal antibody orantigen-binding fragment thereof suspected of being able to compete withsuch invention antibody compound are added. One of the two molecules islabeled. If the labeled compound and the unlabeled compound bind toseparate and discrete sites on CD47, the labeled compound will bind tothe same level whether or not the suspected competing compound ispresent. However, if the sites of interaction are identical oroverlapping, the unlabeled compound will compete, and the amount oflabeled compound bound to the antigen will be lowered. If the unlabeledcompound is present in excess, very little, if any, labeled compoundwill bind. For purposes of the present invention, competing monoclonalantibodies or antigen-binding fragments thereof are those that decreasethe binding of the present antibody compounds to CD47 by about 50%,about 60%, about 70%, about 80%, about 85%, about 90%, about 95%, orabout 99%. Details of procedures for carrying out such competitionassays are well known in the art and can be found, for example, inHarlow and Lane (1988) Antibodies, A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., pages 567-569, ISBN0-87969-314-2. Such assays can be made quantitative by using purifiedantibodies. A standard curve is established by titrating one antibodyagainst itself, i.e., the same antibody is used for both the label andthe competitor. The capacity of an unlabeled competing monoclonalantibody or antigen-binding fragment thereof to inhibit the binding ofthe labeled molecule to the plate is titrated. The results are plotted,and the concentrations necessary to achieve the desired degree ofbinding inhibition are compared. Whether monoclonal antibodies orantigen-binding fragments thereof that compete with antibody compoundsof the present invention in such competition assays possess the same orsimilar functional properties of the present antibody compounds can bedetermined via these methods in conjunction with the methods describedin Examples 3 and 4, below.

The term “treating” (or “treat” or “treatment”) means slowing,interrupting, arresting, controlling, stopping, reducing, or reversingthe progression or severity of a sign, symptom, disorder, condition, ordisease, but does not necessarily involve a total elimination of alldisease-related signs, symptoms, conditions, or disorders. The term“treating” and the like refer to a therapeutic intervention thatameliorates a sign, symptom, etc., of a disease or pathologicalcondition after it has begun to develop.

Acute events and chronic conditions can be treated. In an acute event,an antibody or antigen binding fragment thereof is administered at theonset of a symptom, disorder, condition, disease, or procedure, and isdiscontinued when the acute event ends, or in the case of organtransplantation to the organ, at the time of organ harvest and/or to thetransplant recipient at the time of organ transplantation. In contrast,a chronic symptom, disorder, condition, or disease is treated over amore protracted time frame.

The term “effective amount” refers to the amount or dose of an antibodycompound of the present invention which, upon single or multiple doseadministration to a patient or organ, provides the desired treatment orprevention. Therapeutically effective amounts of the present antibodycompounds can comprise an amount in the range of from about 0.1 mg/kg toabout 150 mg/kg, more preferably from about 0.1 mg/kg to about 100mg/kg, and even more preferably from about 0.1 mg/kg to about 50 mg/kgper single dose administered to a harvested organ or to a patient. Atherapeutically effective amount for any individual patient can bedetermined by the health care provider by monitoring the effect of theantibody compounds on a biomarker, such as serum biomarkers of injury ofthe treated organ, including but not limited to liver, kidney, lung,intestine, pancreas and heart, changes in pulmonary artery pressures,cell surface CD47 expression in tumor or non-tumor tissues, tumorregression, circulating tumor cells or tumor stem cells, etc. Analysisof the data obtained by these methods permits modification of thetreatment regimen during therapy so that optimal amounts of antibodycompounds of the present invention, whether employed alone or incombination with one another, or in combination with another therapeuticagent, or both, are administered, and so that the duration of treatmentcan be determined as well. In this way, the dosing/treatment regimen canbe modified over the course of therapy so that the lowest amounts ofantibody compounds used alone or in combination that exhibitsatisfactory efficacy are administered, and so that administration ofsuch compounds is continued only so long as is necessary to successfullytreat the patient.

The antibody compounds of the present invention can be used asmedicaments in human and veterinary medicine, administered by a varietyof routes. Veterinary applications include the treatment ofcompanion/pet animals, such as cats and dogs; working animals, such asguide or service dogs, and horses; sport animals, such as horses anddogs; zoo animals, such as primates, cats such as lions and tigers,bears, etc.; and other valuable animals kept in captivity.

Most preferably, such compositions are for parenteral administration.Such pharmaceutical compositions can be prepared by methods well knownin the art. See, e.g., Remington: The Science and Practice of Pharmacy,21^(st) Edition (2005), Lippincott Williams & Wilkins, Philadelphia,Pa., and comprise one or more antibody compounds disclosed herein, and apharmaceutically or veterinarily acceptable, e.g., physiologicallyacceptable, carrier, diluent, or excipient.

It should be noted that in all of the therapeutic methods disclosed andclaimed herein, the monoclonal antibodies or antigen binding fragmentsthereof, and monoclonal antibodies or antigen binding fragments thereofthat compete with these monoclonal antibodies or antigen bindingfragments thereof of the present invention that bind to CD47, can beused alone, or in any appropriate combinations with one another, toachieve the greatest treatment efficacy.

The term “tumor” refers to all neoplastic cell growth and proliferation,whether malignant or benign, and all pre-cancerous and cancerous cellsand tissues. The terms “cancer”, “cancerous”, and “tumor” are notmutually exclusive as used herein.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byaberrant cell growth/proliferation. Examples of cancers include, but arenot limited to, carcinomas, lymphomas, blastomas, sarcomas, andleukemias.

The term “susceptible cancer” as used herein refers to a cancer, cellsof which express CD47 and that are responsive to treatment with anantibody or antigen binding fragment thereof of the present invention.Exemplary susceptible cancers include, but are not limited to,leukemias, including acute lymphocytic (lymphoblastic) leukemia, acutemyeloid leukemia, chronic lymphocytic leukemia, multiple myeloma,chronic myeloid leukemia, lymphomas, ovarian cancer, breast cancer,endometrial cancer, colon cancer, rectal cancer, bladder cancer, lungcancer, bronchial cancer, bone cancer, prostate cancer, pancreaticcancer, liver and bile duct cancer, esophageal cancer, renal cancer,thyroid cancer, head and neck cancer, testicular cancer, glioblastoma,astrocytoma, melanoma, and leiomyosarcoma.

The term “directly toxic” refers to the ability of certain of thehumanized antibodies or antigen binding fragments thereof disclosedherein to kill transformed/cancer cells via a cell autonomous mechanismwithout participation of complement or other cells, including but notlimited to, T cells, neutrophils, natural killer cells, macrophages, ordendritic cells.

“Ischemia” refers to a vascular phenomenon in which a decrease in theblood supply to a bodily organ, tissue, or part is caused, for instance,by constriction or obstruction of one or more blood vessels. Ischemiasometimes results from vasoconstriction or thrombosis or embolism.Ischemia can lead to direct ischemic injury, tissue damage due to celldeath caused by reduced oxygen supply. Ischemia can occur acutely, asduring surgery, or from trauma to tissue incurred in accidents, injuriesand war settings, or following harvest of organs intended for subsequenttransplantation, for example. It can also occur sub-acutely, as found inatherosclerotic peripheral vascular disease, where progressive narrowingof blood vessels leads to inadequate blood flow to tissues and organs.

When a tissue is subjected to ischemia, a sequence of chemical events isinitiated that may ultimately lead to cellular dysfunction and necrosis.If ischemia is ended by the restoration of blood flow, a second seriesof injurious events ensue, producing additional injury. Thus, wheneverthere is a transient decrease or interruption of blood flow in asubject, the resultant injury involves two components—the direct injuryoccurring during the ischemic interval, and the indirect or reperfusioninjury that follows.

“Ischemic stroke” can be caused by several different kinds of diseases.The most common problem is narrowing of the arteries in the neck orhead. This is most often caused by atherosclerosis, or gradualcholesterol deposition. If the arteries become too narrow, blood cellsmay collect in them and form blood clots (thrombi). These blood clotscan block the artery where they are formed (thrombosis), or can dislodgeand become trapped in arteries closer to the brain (embolism). Cerebralstroke can occur when atherosclerotic plaque separates away partiallyfrom the vessel wall and occludes the flow of blood through the bloodvessel.

“Reperfusion” refers to restoration of blood flow to tissue that isischemic, due to decrease in blood flow. Reperfusion is a procedure fortreating infarction or other ischemia, by enabling viable ischemictissue to recover, thus limiting further necrosis. However, reperfusioncan itself further damage the ischemic tissue, causing reperfusioninjury.

In addition to the immediate injury that occurs during deprivation ofblood flow, “ischemic/reperfusion injury” involves tissue injury thatoccurs after blood flow is restored. Current understanding is that muchof this injury is caused by chemical products, free radicals, and activebiological agents released by the ischemic tissues.

“Nitric oxide donor or precursor” refers to a compound or agent thateither delivers NO, or that can be converted to NO through enzymatic ornon-enzymatic processes. Examples include, but are not limited to, NOgas, isosorbide dinitrite, nitrite, nitroprusside, nitroglycerin,3-Morpholinosydnonimine (SIN-1), S-nitroso-N-acetylpenicillamine (SNAP),Diethylenetriamine/NO (DETA/NO), S-nitrosothiols, and arginine.

CD47 and Ischemia-Reperfusion Injury (IRI)

Following periods of tissue ischemia, the initiation of blood flowcauses damage referred to as “ischemia-reperfusion injury” or IRI. IRIcontributes to poor outcomes in many surgical procedures where IRIoccurs due to the necessity to stop blood flow for a period of time, inmany forms/causes of trauma in which blood flow is interrupted and laterrestored by therapeutic intervention and in procedures required fororgan transplantation, cardio/pulmonary bypass procedures, reattachmentof severed body parts, reconstructive and cosmetic surgeries and othersituations involving stopping and restarting blood flow. Ischemia itselfcauses many physiological changes that, by themselves would eventuallylead to cell and tissue necrosis and death. Reperfusion poses its ownset of damaging events including generation of reactive oxygen species,thrombosis, inflammation and cytokine mediated damage. The pathways thatare limited by the TSP1-CD47 system are precisely those that would be ofmost benefit in combating the damage of IRI. Thus, blocking theTSP1-CD47 pathway, as with the antibodies disclosed herein, will providemore robust functioning of these endogenous protective pathways.

The humanized anti-CD47 antibodies, antigen binding fragments thereof,and competing antibodies and antigen binding fragments thereof, of thepresent invention can be used in the methods disclosed in U.S. Pat. No.8,236,313, the contents of which are herein incorporated by reference intheir entirety.

CD47 and Cancer

CD47 has been identified as a novel therapeutic target in hematologiccancers (Majeti et al. (2009) Cell 138(2):286-99, as well as in solidtumors such as colon, prostate, breast, and brain cancers (Willingham etal. (2012) Proc Natl Acad Sci USA 109(17):6662-7. Many human cancersup-regulate cell surface expression of CD47 and those expressing thehighest levels of CD47 are the most aggressive and the most lethal forpatients. Increased CD47 expression is thought to protect cancer cellsfrom phagocytic clearance by sending a “don't eat me” signal tomacrophages via SIRPalpha, an inhibitory receptor that preventsphagocytosis of CD47-bearing cells (Jaiswal et al. (2009) Cell138(2):271-851; Chao et al. (2010) Science Translational Medicine2(63):63ra94). Thus, the increase of CD47 expression by many cancersprovides them with a cloak of “selfness” that slows their phagocyticclearance by macrophages and dendritic cells. Anti-CD47 mAbs (CD47 mAbs)that block the CD47/SIRPalpha interaction enhance phagocytosis of cancercells in vitro and contribute to control of tumor burden in publishedhuman to mouse xenograft tumor models. However, there are mechanisms bywhich CD47 mAbs can attack transformed cells that have not yet beenexploited in the war on cancer.

Frazier et al. have shown that a particular anti-human CD47 mAb (clone1F7) has a direct, tumor-toxic effect on human T cell leukemias (Mannaand Frazier (2003) A. J. Immunol. 170:3544-53) and several breastcancers (Manna and Frazier (2004) A. Cancer Research 64(3):1026-36).Other groups have reported such findings in additional types of leukemia(Uno et al. (2007) Oncol. Rep. 17(5):1189-94; Mateo et al. (1999) Nat.Med. 5:1277-84). MAb 1F7 kills CD47 bearing tumor cells without theaction of complement or cell mediated killing by NK cells, T cells ormacrophages. Instead, mAb 1F7 acts via a non-apoptotic mechanism thatinvolves a direct CD47-dependent attack on mitochondria, dischargingtheir membrane potential and destroying the ATP-generating capacity ofthe cell leading to rapid cell death. It is noteworthy that mAb 1F7 doesnot kill resting leukocytes, which also express CD47, but only thosecells that are “activated” by transformation. Thus, normal circulatingcells, all of which express CD47, are spared while cancer cells areselectively killed by the tumor-toxic CD47 mAb (Manna and Frazier (2003)A. J. Immunol. 170:3544-53). This mechanism can be thought of as aproactive, selective and direct attack on tumor cells in contrast to thepassive mechanism of promoting phagocytosis by simply blockingCD47/SIRPalpha binding. Importantly, mAb 1F7 also blocks binding ofSIRPalpha to CD47 and thus it can act via two mechanisms: (1) directtumor cytotoxicity, and (2) promoting phagocytosis of the dead and dyingtumor cells. A single mAb that can accomplish both functions may besuperior to one that only blocks CD47/SIRPalpha binding. In fact, it hasbeen shown that combining a blocking CD47 mAb to promote phagocytosiswith the cytotoxic anti-CD20 mAb, rituximab, is more effective thaneither mAb alone at eradicating human non-Hodgkins lymphoma in axenograft mouse model (Chao et al. (2010) Cell 142(5):699-713). However,rituximab kills by lysing cancer cells, leading to a harsh side effectprofile (Hansel et al. (2010) Nat Rev Drug Discov. 9(4):325-38). Incontrast, the tumor-toxic mAb 1F7 does not cause rapid cell lysis, butrather causes display of phosphatidylserine on the cell surface, thuspromoting phagocytic clearance by this mechanism as well.

Therapeutic Indications

IRI-Related and Autoimmune/Inflammatory Conditions

Administration of a CD47 mAb or antigen binding fragment thereofdisclosed herein can be used to treat a number of diseases andconditions in which IRI is a contributing feature, and to treat variousautoimmune and inflammatory diseases. These include: organtransplantation in which a mAb or antigen binding fragment thereof ofthe present invention is administered to the donor prior to organharvest, to the harvested donor organ, to the organ preservationsolution, to the recipient patient, or to any combination thereof; skingrafting; surgical resections or tissue reconstruction in which such mAbor fragment is administered either locally by injection to the affectedtissue or parenterally to the patient; reattachment of body parts;treatment of traumatic injury; pulmonary hypertension; sickle celldisease (crisis); myocardial infarction; stroke; surgically-inducedischemia; acute kidney disease/kidney failure; any other condition inwhich IRI occurs and contributes to the pathogenesis of disease; andautoimmune/inflammatory diseases, including arthritis, multiplesclerosis, psoriasis, Crohn's disease, inflammatory bowel disease,lupus, Grave's disease and Hashimoto's thyroiditis, and ankylosingspondylitis.

CD47 mAbs and antigen binding fragments thereof of the present inventioncan also be used to increase tissue perfusion in a subject in need ofsuch treatment. Such subjects can be identified by diagnostic proceduresindicating a need for increased tissue perfusion. In addition, the needfor increased tissue perfusion may arise because the subject has had, ishaving, or will have, a surgery selected from integument surgery, softtissue surgery, composite tissue surgery, skin graft surgery, resectionof a solid organ, organ transplant surgery, or reattachment or anappendage or other body part.

Susceptible Cancers

Presently disclosed mAbs effective as cancer therapeutics can beadministered to patients, preferably parenterally, with susceptiblehematologic cancers and solid tumors including, but not limited to,leukemias, including acute lymphocytic (lymphoblastic) leukemia, acutemyeloid leukemia, chronic lymphocytic leukemia, multiple myeloma,chronic myeloid leukemia, lymphomas, ovarian cancer, breast cancer,endometrial cancer, colon cancer, rectal cancer, bladder cancer, lungcancer, bronchial cancer, bone cancer, prostate cancer, pancreaticcancer, liver and bile duct cancer, esophageal cancer, renal cancer,thyroid cancer, head and neck cancer, testicular cancer, glioblastoma,astrocytoma, melanoma, and leiomyosarcoma.

In certain cases, it may be advantageous to administer the mAb directlyto the cancer by injection into the tumor. Since CD47 expression isup-regulated on many cancers, it may also be desirable to use one ormore of the disclosed mAbs as imaging and diagnostic agents when labeledwith radioactive or other tracers known to those skilled in the art ofin vivo imaging of cancers/tumors.

The following examples illustrate various aspects of the presentinvention, but should not be considered as limiting the invention onlyto these particularly disclosed embodiments.

EXAMPLE 1 Production of CD47 Antibodies

The humanized antibodies disclosed herein comprise frameworks derivedfrom the human genome. The collection covers the diversity found in thehuman germ line sequences, yielding functionally expressed antibodies invivo. The complementarity determining regions (CDRs) in the light andheavy chain variable regions of the target chimeric, non-human antibodyVxP037-01LC/VxP037-01HC (SEQ ID NO:7/SEQ ID NO:57) are determinedfollowing commonly accepted rules disclosed, for example, in “ProteinSequence and Structure Analysis of Antibody Variable Domains”, In:Antibody Engineering Lab Manual, Eds. S. Duebel and R. Kontermann,Springer-Verlag, Heidelberg (2001)). The CDR fragments are synthesizedand combined with pools of frameworks to generate full length variabledomains. The humanized variable domains are then combined with asecretion signal and human kappa and human IgG1 constant domains, andcloned into a mammalian expression system (e.g., OptiCHO System,Lifetechnologies, Carlsbad, Calif.) to generate a library of humanizedIgG1 variants. An aliquot of the library is sequenced to ensure highdiversity and integrity of the reading frames of the individual clones.Aliquots of the humanized variant library are then re-arrayed as singleclones into 96 well plates, mini-prepped (e.g., 96 well Miniprep Kit,Qiagen Hilden, Germany), and transfected into CHO cells (Lipofectaminetransfection protocol as recommended by Lifetechnologies, Carlsbad,Calif.). Transfected CHO cells are grown in DMEM medium with 10% FBS(both from Lifetechnologies, Carlsbad, Calif.) at 37° C. under 5% CO₂.The humanized variants are expressed as full length IgG1 molecules, andsecreted into the medium.

The cell culture supernatant containing the humanized IgG variants isthen screened for binding to the target antigen. In parallel, theconcentration of each variant is determined in order to calculatespecific activity for each clone. The specific activity of each clone iscompared to the specific activity of chimeric cloneVxP037-01LC/VxP037-01HC (SEQ ID NO:7/SEQ ID NO:57) expressed on the sameplate, and normalized. Top hits from each plate are re-arrayed andre-screened for confirmation. The final candidates are selected byspecific activity, functional activity, expression level, and sequencediversity, as well as other criteria, as described below.

EXAMPLE 2 CD47 Antibody CDRs

The amino acid sequences of the light chain and heavy chain variableregions, the complete light and heavy chains, and the respectiveencoding nucleotide sequences of the foregoing, of the present humanengineered antibodies are listed below in the section entitled “AminoAcid and Nucleic Acid Sequences.”

The light chain and heavy chain CDR amino acid sequences are shown inTables 1 and 2, respectively.

TABLE 1 Light Chain CDRs CDR1 CDR2 CDR3 RSSQSLVHSNGNTYLH KVSYRFSSQNTHVPRT (SEQ ID NO: 1) (SEQ ID NO: 2) (SEQ ID NO: 3)

TABLE 2 Heavy Chain CDRs CDR1 CDR2 CDR3 GYTFTNYYVF DINPVNGDTNFNEKFKNGGYTMDY (SEQ ID NO: 4) (SEQ ID NO: 5) (SEQ ID NO: 6)

EXAMPLE 3 Binding of Antibodies to CD47 of Different Species

Cross species reactivity of humanized antibodies of the presentinvention is determined using freshly isolated red blood cells (RBCs),which display CD47 on their surface, from human, mouse, rat, and pigaccording to the methods disclosed in Kamel et al. (2010) Blood.Transfus. 8(4):260-266.

Supernatants containing secreted antibodies are collected from CHO cellstransiently transfected with plasmids encoding antibody clones.Transfected CHO cells are grown in F-12 medium containing 10% heatinactivated fetal bovine serum (BioWest; S01520). Antibody concentrationin the supernatants is determined utilizing a quantitative ELISA. ELISAplates are coated with a donkey anti-human FC antibody (Sigma; Catalog#12136) at 10 μg/ml overnight at 4° C. (Promega; Catalog # W4031).Plates are washed with PBS, and then blocked with casein blockingsolution (ThermoScientific; Catalog #37532) for 60 minutes at roomtemperature. Plates are again washed with PBS, tissue culturesupernatants are added, and the plates are incubated for 60 minutes atroom temperature. Plates are then washed three times with PBS andincubated with peroxidase-conjugated goat anti-human IgG (JacksonImmunoresearch Labs; Catalog #109-035-003) for 60 minutes at roomtemperature. Plates are washed three times with PBS, and the peroxidasesubstrate 3,3′,5,5′-tetramethylbenzidine is added (Sigma; Catalog#T4444). Reactions are terminated by the addition of HCl to 0.7N, andabsorbance at 450 nM is determined using a Tecan model Infinite M200plate reader.

RBCs are incubated for 60 minutes on ice with tissue culturesupernatants containing the secreted humanized antibodies at aconcentration of 10 ng/ml in a solution of phosphate buffered saline, pH7.2, 2.5 mM EDTA (PBS+E). Cells are then washed with cold PBS+E, andincubated for an additional hour on ice with FITC labeled donkeyanti-human antibody (Jackson Immuno Research Labs, West Grove, Pa.;Catalogue #709-096-149) in PBS+E. Cells are then washed with PBS+E, andantibody binding is analyzed using a BD FACSAria Cell Sorter (BectonDickinson) or a C6 Accuri Flow Cytometer (Becton Dickinson). Antibodybinding is quantitated by comparison of mean fluorescence valuesrelative to that of chimeric antibody >VxP037-01LC (SEQ IDNO:7))/>VxP037-01HC (SEQ ID NO:57). The mean fluorescence value for eachantibody is divided by the mean fluorescence value for the chimericantibody.

The results are shown in Table 3, where “Chimera” represents chimericantibody >VxP037-01LC (SEQ ID NO:7))/>VxP037-01HC (SEQ ID NO:57), Clone1 represents >pVxK7b-037-hum01-LC (SEQ ID NO:8)/>pVxK7b-037-hum01-HC(SEQ ID NO:58), Clone 2 represents >pVxK7b-037-hum02-LC (SEQ IDNO:9)/>pVxK7b-037-hum02-HC (SEQ ID NO:59), and so on similarly forremaining clones 3-24.

TABLE 3 Binding of Humanized Antibodies to CD47 on the Surface of RedBlood Cells of Different Mammalian Species Clone No. Human Mouse Rat PigChimera 1.0 1.0 1.0 1.0 1 1.1 1.7 2.7 1.3 2 1.0 1.2 2.6 1.2 3 0.7 0.91.7 0.9 4 0.6 0.6 1.0 0.6 5 1.0 1.0 2.2 1.2 6 0.9 1.2 2.1 1.1 7 0.5 0.40.8 0.9 8 0.7 0.7 1.2 0.8 9 1.2 1.4 3.7 1.6 10 1.1 1.2 2.9 1.5 11 0.80.7 1.2 1.2 12 0.8 0.6 1.3 1.4 13 1.2 1.3 3.1 1.4 14 1.1 1.5 3.2 1.4 151.0 1.3 2.4 1.2 16 0.9 1.0 2.1 1.1 17 0.8 0.9 2.1 1.3 18 1.0 1.3 2.2 1.219 0.7 1.0 2.6 1.3 20 1.3 1.5 1.9 1.7 21 1.2 1.2 2.8 1.4 22 1.1 1.2 2.81.4 23 1.2 1.4 3.3 1.7 24 0.8 0.7 1.2 1.1

These data demonstrate that all of the humanized CD47 mAb clonesdisclosed herein bind well to CD47 of a variety of different mammalianspecies, confirming the useful cross-species reactivity of theseantibodies.

EXAMPLE 4 Cell Viability Assay

The purpose of this experiment is to identify antibody clones of thepresent invention that do, and do not, exhibit cytotoxic activity. Foruse in cardiovascular indications, including transplantation and otherapplications related to IRI, the therapeutic mAb should lack cytotoxicactivity. In contrast, antibodies useful in the treatment of cancershould exhibit toxicity against transformed/cancer cells. Thisadditional property of selective toxicity to cancer cells is expected tohave advantages compared to mAbs that only prevent SIRPalpha binding toCD47.

The method employed is described in Vistica et al. (1991) Cancer Res.51:2515-2520.

Jurkat JE6.1 cells (ATCC, Manassas, Va.; Catalog # TIB-152) are grown inIscove's modified Dulbeccco's medium containing 5% (v/v) heatinactivated fetal bovine serum (BioWest; Catalogue # S01520), 100units/mL penicillin, 100 μg mL streptomycin (Sigma; Catalogue # P4222)at densities less than 1×10⁶ cells/mL. For the cell viability assay,cells are plated in 96 well tissue culture plates at a density of 2×10⁴cells/ml in Iscoves modified Dulbecco's medium containing 5% (v/v) heatinactivated fetal bovine serum (BioWest; Catalog # S01520), 100 units/mLpenicillin, 100 μg/mL streptomycin (Sigma; #P4222) along with humanizedantibodies as disclosed herein at a final concentration of 10 ng/ml,prepared as described above in Example 3. Cells are incubated for 72hours at 37° C. in an atmosphere of 5% (v/v) CO₂. Cell density is thenquantitated using WST1 reagent (Roche Applied Science, Indianapolis,Ind.; Catalog #05015944001) according to the manufacturer'sinstructions. The effect of the antibodies on cell growth is quantitatedby comparison to growth of cells containing no added antibody (PBS;average percent killing=0).

The results are shown in table 4. The values in the table represent themean of 3 separate experiments. “Chimera” and clone numbers are asdescribed above in Example 3. 1F7 is the anti-human CD47 mAb, discussedabove, that has a direct, tumor-toxic effect on human T cell leukemias(Manna and Frazier (2003) A. J. Immunol. 170:3544-53) and several breastcancers (Manna and Frazier (2004) A. Cancer Research 64(3):1026-36).

TABLE 4 Cytotoxicity of Humanized CD47mAbs on Transformed Human T Cells,Jurkat JE6.1 Clone No. Average % Killing % of 1F7 Cytotoxic Chimera 4.319  1 −3.3 −14  2 9.8 43 Yes  3 8.6 38 Yes  4 6.8 30  5 11.8 52 Yes  614 61 Yes  7 1.8 8  8 10.6 46 Yes  9 1 4 10 7.4 32 Yes 11 −7.2 −32 12−6.9 −30 13 17.8 78 Yes 14 16.5 72 Yes 15 8.1 36 Yes 16 8.7 38 Yes 1712.4 54 Yes 18 5.4 23 19 9.6 42 Yes 20 3.1 14 21 4.5 20 22 −0.7 −3 234.8 21 24 −13.1 −57 1F7 22.9 100 Yes

These data demonstrate that the majority of the present humanizedantibody clones are not significantly cytotoxic toward Jurkat T cells.However, certain of the clones have significant cytotoxicity, similar topreviously identified mouse anti-human CD47 mAb 1F7 (Manna and Frazier,J. Immunol. (2003) 170(7):3544-53.

The following clones, indicated in Table 2 with a “Yes”, are consideredto be cytotoxic: 2, 3, 5, 6, 8, 10, 13, 14, 15, 16, 17, and 19.

The following clones are considered to be non-toxic: 1, 4, 7, 9, 11, 12,18, 20, 21, 22, 23, and 24.

The invention being thus described, it will be obvious that the same maybe varied in many ways. Such variations are not to be regarded as adeparture from the spirit and scope of the invention, and all suchmodifications as would be obvious to one skilled in the art are intendedto be included within the scope of the following claims.

Amino Acid and Nucleic Acid Sequences

Light Chain Variable Region Amino Acid Sequences

>VxP037-01LC: Underlined amino acid sequences represent CDRs(SEQ ID NO: 7) DVVMTQTPLSLSVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSYRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQNTHVPRTFGQG  >pVxK7b-037-hum01-LC  (SEQ ID NO: 8)DIVMTQTPLSLPVTPGEPASISCRSSQSLVHSNGNTYLHWYQQKPGKAPKLLIYKVSYRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPRTFGQG   >pVxK7b-037-hum02-LC  (SEQ ID NO: 9) DVVMTQSPLSLPVTLGQPASISCRSSQSLVHSNGNTYLHWYQQKPGQAPRLLIYKVSYRFSGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCSQNTHVPRTFGQG >pVxK7b-037-hum03-LC  (SEQ ID NO: 10) DVVMTQSPLSLPVTLGQPASISCRSSQSLVHSNGNTYLHWYQQKPGKAPKLLIYKVSYRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPRTFGQG  >pVxK7b-037-hum04-LC  (SEQ ID NO: 11) DIQMTQSPSSLSASVGDRVTITCRSSQSLVHSNGNTYLHWYLQKPGQSPQLLIYKVSYRFGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCSQNTHVPRTFGQG >pVxK7b-037-hum05-LC  (SEQ ID NO: 12)DIVMTQTPLSLPVTPGEPASISCRSSQSLVHSNGNTYLHWYLQKPGQSPQ LLIYKVSYRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVP  RTFGQG >pVxK7b-037-hum06-LC  (SEQ ID NO: 13)DIQMTQSPSSLSASVGDRVTITCRSSQSLVHSNGNTYLHWYLQKPGQSPQ LLIYKVSYRFSGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCSQNTHVP  RTFGQG >pVxK7b-037-hum07-LC  (SEQ ID NO: 14) DIVMTQTPLSLPVTPGEPASISCRSSQSLVHSNGNTYLHWYLQKPGQSPQ LLIYKVSYRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPRTFGQG  >pVxK7b-037-hum08-LC  (SEQ ID NO: 15) DIVMTQTPLSLPVTPGEPASISCRSSQSLVHSNGNTYLHWYQQKPGKAPK LLIYKVSYRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVP RTFGQG >pVxK7b-037-hum09-LC  (SEQ ID NO: 16) DVVMTQSPLSLPVTLGQPASISCRSSQSLVHSNGNTYLHWYQQKPGKAPK LLIYKVSYRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVP RTFGQG >pVxK7b-037-hum10-LC  (SEQ ID NO: 17) DIVMTQTPLSLPVTPGEPASISCRSSQSLVHSNGNTYLHWYLQKPGQSPQLLIYKVSYRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVP RTFGQG >pVxK7b-037-humll-LC  (SEQ ID NO: 18) EIVLTQSPATLSVSPGERATLSCRSSQSLVHSNGNTYLHWYQQKPGQAPRLLIYKVSYRFSGVPSRFSGSGSGTDFTFTISSLEAEDAATYYCSQNTHVP RTFGQG >pVxK7b-037-hum12-LC  (SEQ ID NO: 19) DIVMTQTPLSLPVTPGEPASISCRSSQSLVHSNGNTYLHWYQQKPGKAPK LLIYKVSYRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVP RTFGQG >pVxK7b-037-hum13-LC  (SEQ ID NO: 20) DIVMTQTPLSLPVTPGEPASISCRSSQSLVHSNGNTYLHWYQQKPGKAPK LLIYKVSYRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVP RTFGQG >pVxK7b-037-hum14-LC  (SEQ ID NO: 21)DVVMTQSPLSLPVTLGQPASISCRSSQSLVHSNGNTYLHWYQQKPGKAPK LLIYKVSYRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVP  RTFGQG >pVxK7b-037-hum15-LC  (SEQ ID NO: 22) AIQLTQSPSSLSASVGDRVTITCRSSQSLVHSNGNTYLHWYQQKPGQAPR LLIYKVSYRFSGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCSQNTHVP RTFGQG >pVxK7b-037-hum16-LC  (SEQ ID NO: 23) AIQLTQSPSSLSASVGDRVTITCRSSQSLVHSNGNTYLHWYQQKPGQAPR LLIYKVSYRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVP RTFGQG >pVxK7b-037-hum17-LC  (SEQ ID NO: 24)AIQLTQSPSSLSASVGDRVTITCRSSQSLVHSNGNTYLHWYQQKPGQAPR LLIYKVSYRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVP  RTFGQG >pVxK7b-037-hum18-LC  (SEQ ID NO: 25)EIVLTQSPATLSVSPGERATLSCRSSQSLVHSNGNTYLHWYQQKPGQAPR LLIYKVSYRFSGVPSRFSGSGSGTDFTFTISSLEAEDAATYYCSQNTHVP  RTFGQG >pVxK7b-037-hum19-LC  (SEQ ID NO: 26) DVVMTQSPLSLPVTLGQPASISCRSSQSLVHSNGNTYLHWYQQKPGKAPK LLIYKVSYRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVP RTFGQG >pVxK7b-037-hum20-LC  (SEQ ID NO: 27)DIVMTQTPLSLPVTPGEPASISCRSSQSLVHSNGNTYLHWYLQKPGQSPQ LLIYKVSYRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVP  RTFGQG >pVxK7b-037-hum21-LC  (SEQ ID NO: 28) AIQLTQSPSSLSASVGDRVTITCRSSQSLVHSNGNTYLHWYQQKPGQAPR LLIYKVSYRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVP RTFGQG >pVxK7b-037-hum22-LC  (SEQ ID NO: 29)EIVLTQSPATLSVSPGERATLSCRSSQSLVHSNGNTYLHWYQQKPGQAPRLLIYKVSYRFSGVPSRFSGSGSGTDFTFTISSLEAEDAATYYCSQNTHVP  RTFGQG >pVxK7b-037-hum23-LC  (SEQ ID NO: 30)DIVMTQTPLSLPVTPGEPASISCRSSQSLVHSNGNTYLHWYQQKPGKAPK LLIYKVSYRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVP  RTFGQG >pVxK7b-037-hum24-LC  (SEQ ID NO: 31) AIQLTQSPSSLSASVGDRVTITCRSSQSLVHSNGNTYLHWYQQKPGQAPR LLIYKVSYRFSGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCSQNTHVP  RTFGQGLight Chain Variable Region Nucleic Acid Sequences

>VxP037-01LC (SEQ ID NO: 32)GATGTTGTTATGACCCAAACTCCACTCTCCCTGTCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCCTTGTACACAGTAATGGAAACACCTATTTACATTGGTACCTGCAGAAGCCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCTACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTTCTGCTCTCAAAATACACATGTTCCTCGGACGTTCGGCCAAGGAG >pVxK7b-037-hum01-LC (SEQ ID NO: 33)GATATTGTGATGACCCAGACTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGATCTAGTCAGAGCCTTGTACACAGTAATGGAAACACCTATTTACATTGGTATCAGCAGAAACCAGGGAAAGCTCCTAAGCTCCTGATCTATAAAGTTTCCTACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGGTCAGGCACTGATTTCACACTGAAAATCAGCAGGGTGGAGGCTGAGGATGTTGGAGTTTATTACTGTTCTCAAAATACACATGTTCCTCGGACGTTCGGCCAAGGG >pVxK7b-037-hum02-LC (SEQ ID NO: 34)GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCTTGGACAGCCGGCCTCCATCTCCTGCAGATCTAGTCAGAGCCTTGTACACAGTAATGGAAACACCTATTTACATTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATAAAGTTTCCTACCGATTTTCTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGTTCTCAAAATACACATGTTCCTCGGACGTTCGGCCAAGGG >pVxK7b-037-hum03-LC (SEQ ID NO: 35)GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCTTGGACAGCCGGCCTCCATCTCCTGCAGATCTAGTCAGAGCCTTGTACACAGTAATGGAAACACCTATTTACATTGGTATCAGCAGAAACCAGGGAAAGCTCCTAAGCTCCTGATCTATAAAGTTTCCTACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGGTCAGGCACTGATTTCACACTGAAAATCAGCAGGGTGGAGGCTGAGGATGTTGGAGTTTATTACTGTTCTCAAAATACACATGTTCCTCGGACGTTCGGCCAAGGG >pVxK7b-037-hum04-LC (SEQ ID NO: 36)GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCAGATCTAGTCAGAGCCTTGTACACAGTAATGGAAACACCTATTTACATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATAAAGTTTCCTACCGATTTTCTGGGATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGAGTTCACTCTCACCATCAGCAGCCTGCAGTCTGAAGATTTTGCAGTTTATTACTGTTCTCAAAATACACATGTTCCTCGGACGTTCGGCCAAGGG >pVxK7b-037-hum05-LC (SEQ ID NO: 37)GATATTGTGATGACCCAGACTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGATCTAGTCAGAGCCTTGTACACAGTAATGGAAACACCTATTTACATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATAAAGTTTCCTACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGGTCAGGCACTGATTTCACACTGAAAATCAGCAGGGTGGAGGCTGAGGATGTTGGAGTTTATTACTGTTCTCAAAATACACATGTTCCTCGGACGTTCGGCCAAGGG >pVxK7b-037-hum06-LC (SEQ ID NO: 38)GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCAGATCTAGTCAGAGCCTTGTACACAGTAATGGAAACACCTATTTACATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATAAAGTTTCCTACCGATTTTCTGGGATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGAGTTCACTCTCACCATCAGCAGCCTGCAGTCTGAAGATTTTGCAGTTTATTACTGTTCTCAAAATACACATGTTCCTCGGACGTTCGGCCAAGGG >pVxK7b-037-hum07-LC (SEQ ID NO: 39)GATATTGTGATGACCCAGACTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGATCTAGTCAGAGCCTTGTACACAGTAATGGAAACACCTATTTACATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATAAAGTTTCCTACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGGTCAGGCACTGATTTCACACTGAAAATCAGCAGGGTGGAGGCTGAGGATGTTGGAGTTTATTACTGTTCTCAAAATACACATGTTCCTCGGACGTTCGGCCAAGGG >pVxK7b-037-hum08-LC (SEQ ID NO: 40)GATATTGTGATGACCCAGACTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGATCTAGTCAGAGCCTTGTACACAGTAATGGAAACACCTATTTACATTGGTATCAGCAGAAACCAGGGAAAGCTCCTAAGCTCCTGATCTATAAAGTTTCCTACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGGTCAGGCACTGATTTCACACTGAAAATCAGCAGGGTGGAGGCTGAGGATGTTGGAGTTTATTACTGTTCTCAAAATACACATGTTCCTCGGACGTTCGGCCAAGGG >pVxK7b-037-hum09-LC (SEQ ID NO: 41)GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCTTGGACAGCCGGCCTCCATCTCCTGCAGATCTAGTCAGAGCCTTGTACACAGTAATGGAAACACCTATTTACATTGGTATCAGCAGAAACCAGGGAAAGCTCCTAAGCTCCTGATCTATAAAGTTTCCTACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGGTCAGGCACTGATTTCACACTGAAAATCAGCAGGGTGGAGGCTGAGGATGTTGGAGTTTATTACTGTTCTCAAAATACACATGTTCCTCGGACGTTCGGCCAAGGG >pVxK7b-037-hum10-LC (SEQ ID NO: 42)GATATTGTGATGACCCAGACTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGATCTAGTCAGAGCCTTGTACACAGTAATGGAAACACCTATTTACATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATAAAGTTTCCTACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGGTCAGGCACTGATTTCACACTGAAAATCAGCAGGGTGGAGGCTGAGGATGTTGGAGTTTATTACTGTTCTCAAAATACACATGTTCCTCGGACGTTCGGCCAAGGG >pVxK7b-037-hum11-LC (SEQ ID NO: 43)GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGATCTAGTCAGAGCCTTGTACACAGTAATGGAAACACCTATTTACATTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATAAAGTTTCCTACCGATTTTCTGGGGTCCCCTCGAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACCTTTACCATCAGTAGCCTGGAAGCTGAAGATGCTGCAACATATTACTGTTCTCAAAATACACATGTTCCTCGGACGTTCGGCCAAGGG >pVxK7b-037-hum12-LC (SEQ ID NO: 44)GATATTGTGATGACCCAGACTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGATCTAGTCAGAGCCTTGTACACAGTAATGGAAACACCTATTTACATTGGTATCAGCAGAAACCAGGGAAAGCTCCTAAGCTCCTGATCTATAAAGTTTCCTACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGGTCAGGCACTGATTTCACACTGAAAATCAGCAGGGTGGAGGCTGAGGATGTTGGAGTTTATTACTGTTCTCAAAATACACATGTTCCTCGGACGTTCGGCCAAGGG >pVxK7b-037-hum13-LC (SEQ ID NO: 45)GATATTGTGATGACCCAGACTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGATCTAGTCAGAGCCTTGTACACAGTAATGGAAACACCTATTTACATTGGTATCAGCAGAAACCAGGGAAAGCTCCTAAGCTCCTGATCTATAAAGTTTCCTACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGGTCAGGCACTGATTTCACACTGAAAATCAGCAGGGTGGAGGCTGAGGATGTTGGAGTTTATTACTGTTCTCAAAATACACATGTTCCTCGGACGTTCGGCCAAGGG >pVxK7b-037-hum14-LC (SEQ ID NO: 46)GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCTTGGACAGCCGGCCTCCATCTCCTGCAGATCTAGTCAGAGCCTTGTACACAGTAATGGAAACACCTATTTACATTGGTATCAGCAGAAACCAGGGAAAGCTCCTAAGCTCCTGATCTATAAAGTTTCCTACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGGTCAGGCACTGATTTCACACTGAAAATCAGCAGGGTGGAGGCTGAGGATGTTGGAGTTTATTACTGTTCTCAAAATACACATGTTCCTCGGACGTTCGGCCAAGGG >pVxK7b-037-hum15-LC (SEQ ID NO: 47)GCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCAGATCTAGTCAGAGCCTTGTACACAGTAATGGAAACACCTATTTACATTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATAAAGTTTCCTACCGATTTTCTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGTTCTCAAAATACACATGTTCCTCGGACGTTCGGCCAAGGG >pVxK7b-037-hum16-LC (SEQ ID NO: 48)GCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCAGATCTAGTCAGAGCCTTGTACACAGTAATGGAAACACCTATTTACATTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATAAAGTTTCCTACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGGTCAGGCACTGATTTCACACTGAAAATCAGCAGGGTGGAGGCTGAGGATGTTGGAGTTTATTACTGTTCTCAAAATACACATGTTCCTCGGACGTTCGGCCAAGGG >pVxK7b-037-hum17-LC (SEQ ID NO: 49)GCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCAGATCTAGTCAGAGCCTTGTACACAGTAATGGAAACACCTATTTACATTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATAAAGTTTCCTACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGGTCAGGCACTGATTTCACACTGAAAATCAGCAGGGTGGAGGCTGAGGATGTTGGAGTTTATTACTGTTCTCAAAATACACATGTTCCTCGGACGTTCGGCCAAGGG >pVxK7b-037-hum18-LC (SEQ ID NO: 50)GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGATCTAGTCAGAGCCTTGTACACAGTAATGGAAACACCTATTTACATTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATAAAGTTTCCTACCGATTTTCTGGGGTCCCCTCGAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACCTTTACCATCAGTAGCCTGGAAGCTGAAGATGCTGCAACATATTACTGTTCTCAAAATACACATGTTCCTCGGACGTTCGGCCAAGGG >pVxK7b-037-hum19-LC (SEQ ID NO: 51)GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCTTGGACAGCCGGCCTCCATCTCCTGCAGATCTAGTCAGAGCCTTGTACACAGTAATGGAAACACCTATTTACATTGGTATCAGCAGAAACCAGGGAAAGCTCCTAAGCTCCTGATCTATAAAGTTTCCTACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGGTCAGGCACTGATTTCACACTGAAAATCAGCAGGGTGGAGGCTGAGGATGTTGGAGTTTATTACTGTTCTCAAAATACACATGTTCCTCGGACGTTCGGCCAAGGG >pVxK7b-037-hum20-LC (SEQ ID NO: 52)GATATTGTGATGACCCAGACTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGATCTAGTCAGAGCCTTGTACACAGTAATGGAAACACCTATTTACATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATAAAGTTTCCTACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGGTCAGGCACTGATTTCACACTGAAAATCAGCAGGGTGGAGGCTGAGGATGTTGGAGTTTATTACTGTTCTCAAAATACACATGTTCCTCGGACGTTCGGCCAAGGG >pVxK7b-037-hum21-LC (SEQ ID NO: 53)GCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCAGATCTAGTCAGAGCCTTGTACACAGTAATGGAAACACCTATTTACATTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATAAAGTTTCCTACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGGTCAGGCACTGATTTCACACTGAAAATCAGCAGGGTGGAGGCTGAGGATGTTGGAGTTTATTACTGTTCTCAAAATACACATGTTCCTCGGACGTTCGGCCAAGGG >pVxK7b-037-hum22-LC (SEQ ID NO: 54)GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGATCTAGTCAGAGCCTTGTACACAGTAATGGAAACACCTATTTACATTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATAAAGTTTCCTACCGATTTTCTGGGGTCCCCTCGAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACCTTTACCATCAGTAGCCTGGAAGCTGAAGATGCTGCAACATATTACTGTTCTCAAAATACACATGTTCCTCGGACGTTCGGCCAAGGG >pVxK7b-037-hum23-LC (SEQ ID NO: 55)GATATTGTGATGACCCAGACTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGATCTAGTCAGAGCCTTGTACACAGTAATGGAAACACCTATTTACATTGGTATCAGCAGAAACCAGGGAAAGCTCCTAAGCTCCTGATCTATAAAGTTTCCTACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGGTCAGGCACTGATTTCACACTGAAAATCAGCAGGGTGGAGGCTGAGGATGTTGGAGTTTATTACTGTTCTCAAAATACACATGTTCCTCGGACGTTCGGCCAAGGG >pVxK7b-037-hum24-LC (SEQ ID NO: 56)GCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCAGATCTAGTCAGAGCCTTGTACACAGTAATGGAAACACCTATTTACATTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATAAAGTTTCCTACCGATTTTCTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGTTCTCAAAATACACATGTTCCTCGGACGTTCGGCCAAGGGHeavy Chain Variable Region Amino Acid Sequences

>VxP037-01HC (SEQ ID NO: 57)EVQLQQFGAELVKPGASMKLSCKASGYTFTNYYVFWVKQRPGQGLEWIGDINPVNGDTNFNEKFKNKATLTVDKSSTTTYLQLSSLTSEDSAVYYCTRGGYTMDYWGQG >pVxK7b-037-hum01-HC (SEQ ID NO: 58)QVQLQESGPGLVKPSQTLSLTCTVSGYTFTNYYVFWVRQARGQRLEWIGDINPVNGDTNFNEKFKNRVTISADKSISTAYLQWSSLKASDTAMYYCARGGYTMDYWGQG >pVxK7b-037-hum02-HC (SEQ ID NO: 59)QVQLQESGPGLVKPSQTLSLTCTVSGYTFTNYYVFWVRQARGQRLEWIGDINPVNGDTNFNEKFKNRVTISADKSISTAYLQWSSLKASDTAMYYCARGGYTMDYWGQG >pVxK7b-037-hum03-HC (SEQ ID NO: 60)EVQLVQSGAEVKKPGESLRISCKGSGYTFTNYYVFWVRQARGQRLEWIGDINPVNGDTNFNEKFKNRVTITADKSTSTAYMELSSLRSEDTAVYYCARGGYTMDYWGQG >pVxK7b-037-hum04-HC (SEQ ID NO: 61)QVQLQESGPGLVKPSQTLSLTCTVSGYTFTNYYVFWVRQAPGKGLEWVSDINPVNGDTNFNEKFKNRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGGYTMDYWGQG >pVxK7b-037-hum05-HC (SEQ ID NO: 62)QVQLQESGPGLVKPSQTLSLTCTVSGYTFTNYYVFWVRQAPGKGLEWVSDINPVNGDTNFNEKFKNRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGGYTMDYWGQG >pVxK7b-037-hum06-HC (SEQ ID NO: 63)QVQLQESGPGLVKPSQTLSLTCTVSGYTFTNYYVFWVRQARGQRLEWIGDINPVNGDTNFNEKFKNRLTISKDTSKNQVVLTMTNMDPVDTATYYCARGGYTMDYWGQG >pVxK7b-037-hum07-HC (SEQ ID NO: 64)QVQLQESGPGLVKPGATVKISCKVSGYTFTNYYVFWVRQARGQRLEWIGDINPVNGDTNFNEKFKNRVTITADKSTSTAYMELSSLRSEDTAVYYCARGGYTMDYWGQG >pVxK7b-037-hum08-HC (SEQ ID NO: 65)QITLKESGPTLVKPTQTLTLTCTFSGYTFTNYYVFWIRQSPSRGLEWLGDINPVNGDTNFNEKFKNRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGGYTMDYWGQG >pVxK7b-037-hum09-HC (SEQ ID NO: 66)QVQLQESGPGLVKPSQTLSLTCTVSGYTFTNYYVFWVRQARGQRLEWIGDINPVNGDTNFNEKFKNRLTISKDTSKNQVVLTMTNMDPVDTATYYCARGGYTMDYWGQG >pVxK7b-037-hum10-HC (SEQ ID NO: 67)QVQLQESGPGLVKPSQTLSLTCTVSGYTFTNYYVFWVRQARGQRLEWIGDINPVNGDTNFNEKFKNRLTISKDTSKNQVVLTMTNMDPVDTATYYCARGGYTMDYWGQG >pVxK7b-037-hum11-HC (SEQ ID NO: 68)QVQLQESGPGLVKPGATVKISCKVSGYTFTNYYVFWVRQARGQRLEWIGDINPVNGDTNFNEKFKNRVTITADKSTSTAYMELSSLRSEDTAVYYCARGGYTMDYWGQG >pVxK7b-037-hum12-HC (SEQ ID NO: 69)QVQLQESGPGLVKPGATVKISCKVSGYTFTNYYVFWVRQARGQRLEWIGDINPVNGDTNFNEKFKNRVTITADKSTSTAYMELSSLRSEDTAVYYCARGGYTMDYWGQG >pVxK7b-037-hum13-HC (SEQ ID NO: 70)EVQLVQSGAEVKKPGESLRISCKGSGYTFTNYYVFWIRQSPSRGLEWLGDINPVNGDTNFNEKFKNRVTITADKSTSTAYMELSSLRSEDTAVYYCARGGYTMDYWGQG >pVxK7b-037-hum14-HC (SEQ ID NO: 71)QVQLQESGPGLVKPSQTLSLTCTVSGYTFTNYYVFWVRQARGQRLEWIGDINPVNGDTNFNEKFKNRVTISADKSISTAYLQWSSLKASDTAMYYCARGGYTMDYWGQG >pVxK7b-037-hum15-HC (SEQ ID NO: 72)QVQLQESGPGLVKPSQTLSLTCTVSGYTFTNYYVFWVRQARGQRLEWIGDINPVNGDTNFNEKFKNRLTISKDTSKNQVVLTMTNMDPVDTATYYCARGGYTMDYWGQG >pVxK7b-037-hum16-HC (SEQ ID NO: 73)QVQLQESGPGLVKPSQTLSLTCTVSGYTFTNYYVFWVRQARGQRLEWIGDINPVNGDTNFNEKFKNRVTISADKSISTAYLQWSSLKASDTAMYYCARGGYTMDYWGQG >pVxK7b-037-hum17-HC (SEQ ID NO: 74)EVQLVQSGAEVKKPGATVKISCKVSGYTFTNYYVFWIRQPPGKGLEWIGDINPVNGDTNFNEKFKNRVTITADKSTSTAYMELSSLRSEDTAVYYCARGGYTMDYWGQG >pVxK7b-037-hum18-HC (SEQ ID NO: 75)EVQLVQSGAEVKKPGESLRISCKGSGYTFTNYYVFWIRQSPSRGLEWLGDINPVNGDTNFNEKFKNRVTITADKSTSTAYMELSSLRSEDTAVYYCARGGYTMDYWGQG >pVxK7b-037-hum19-HC (SEQ ID NO: 76)EVQLVQSGAEVKKPGESLRISCKGSGYTFTNYYVFWIRQSPSRGLEWLGDINPVNGDTNFNEKFKNRVTITADKSTSTAYMELSSLRSEDTAVYYCARGGYTMDYWGQG >pVxK7b-037-hum20-HC (SEQ ID NO: 77)QITLKESGPTLVKPTQTLTLTCTFSGYTFTNYYVFWVRQAPGQGLEWMGDINPVNGDTNFNEKFKNRVTITADKSTSTAYMELSSLRSEDTAVYYCARGGYTMDYWGQG >pVxK7b-037-hum21-HC (SEQ ID NO: 78)QVQLQESGPGLVKPSQTLSLTCTVSGYTFTNYYVFWVRQARGQRLEWIGDINPVNGDTNFNEKFKNRLTISKDTSKNQVVLTMTNMDPVDTATYYCARGGYTMDYWGQG >pVxK7b-037-hum22-HC (SEQ ID NO: 79)QVQLQESGPGLVKPSQTLSLTCTVSGYTFTNYYVFWVRQARGQRLEWIGDINPVNGDTNFNEKFKNRLTISKDTSKNQVVLTMTNMDPVDTATYYCARGGYTMDYWGQG >pVxK7b-037-hum23-HC (SEQ ID NO: 80)QVQLQESGPGLVKPSQTLSLTCTVSGYTFTNYYVFWVRQARGQRLEWIGDINPVNGDTNFNEKFKNRLTISKDTSKNQVVLTMTNMDPVDTATYYCARGGYTMDYWGQG >pVxK7b-037-hum24-HC (SEQ ID NO: 81)QVQLQESGPGLVKPGATVKISCKVSGYTFTNYYVFWVRQARGQRLEWIGDINPVNGDTNFNEKFKNRVTITADKSTSTAYMELSSLRSEDTAVYYC ARGGYTMDYWGQGHeavy Chain Variable Region Nucleic Acid Sequences

>VxP037-01HC (SEQ ID NO: 82)GAGGTCCAGCTGCAGCAGTTTGGGGCTGAACTGGTGAAGCCTGGGGCTTCAATGAAGTTGTCCTGCAAGGCTTCTGGCTACACCTTCACCAACTACTATGTATTCTGGGTGAAACAGAGGCCTGGACAAGGCCTTGAGTGGATTGGAGACATTAATCCTGTCAATGGTGATACTAACTTCAATGAGAAATTCAAGAACAAGGCCACACTGACTGTAGACAAGTCCTCCACCACAACATACTTGCAACTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTACAAGAGGGGGTTATACTATGGACTACTGGGGTCAAGGA >pVxK7b-037-hum01-HC(SEQ ID NO: 83) CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGACCCTGTCCCTCACCTGCACTGTCTCTGGCTACACCTTCACCAACTACTATGTATTCTGGGTGCGACAGGCTCGTGGACAACGCCTTGAGTGGATAGGTGACATTAATCCTGTCAATGGTGATACTAACTTCAATGAGAAATTCAAGAACAGAGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTACCTGCAGTGGAGCAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGAGGGGGTTATACTATGGACTACTGGGGCCAGGGA >pVxK7b-037-hum02-HC(SEQ ID NO: 84) CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGACCCTGTCCCTCACCTGCACTGTCTCTGGCTACACCTTCACCAACTACTATGTATTCTGGGTGCGACAGGCTCGTGGACAACGCCTTGAGTGGATAGGTGACATTAATCCTGTCAATGGTGATACTAACTTCAATGAGAAATTCAAGAACAGAGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTACCTGCAGTGGAGCAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGAGGGGGTTATACTATGGACTACTGGGGCCAGGGA >pVxK7b-037-hum03-HC(SEQ ID NO: 85) GAAGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAGGATCTCCTGTAAGGGTTCTGGCTACACCTTCACCAACTACTATGTATTCTGGGTGCGACAGGCTCGTGGACAACGCCTTGAGTGGATAGGTGACATTAATCCTGTCAATGGTGATACTAACTTCAATGAGAAATTCAAGAACAGAGTCACGATTACCGCGGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAGGGGGTTATACTATGGACTACTGGGGCCAGGGA >pVxK7b-037-hum04-HC(SEQ ID NO: 86) CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGACCCTGTCCCTCACCTGCACTGTCTCTGGCTACACCTTCACCAACTACTATGTATTCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCAGTGACATTAATCCTGTCAATGGTGATACTAACTTCAATGAGAAATTCAAGAACAGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCGGACACGGCTGTGTATTACTGTGCGAGAGGGGGTTATACTATGGACTACTGGGGCCAGGGA >pVxK7b-037-hum05-HC(SEQ ID NO: 87) CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGACCCTGTCCCTCACCTGCACTGTCTCTGGCTACACCTTCACCAACTACTATGTATTCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCAGTGACATTAATCCTGTCAATGGTGATACTAACTTCAATGAGAAATTCAAGAACAGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCGGACACGGCTGTGTATTACTGTGCGAGAGGGGGTTATACTATGGACTACTGGGGCCAGGGA >pVxK7b-037-hum06-HC(SEQ ID NO: 88) CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGACCCTGTCCCTCACCTGCACTGTCTCTGGCTACACCTTCACCAACTACTATGTATTCTGGGTGCGACAGGCTCGTGGACAACGCCTTGAGTGGATAGGTGACATTAATCCTGTCAATGGTGATACTAACTTCAATGAGAAATTCAAGAACAGACTCACCATCTCCAAGGACACCTCCAAAAACCAGGTGGTCCTTACAATGACCAACATGGACCCTGTGGACACAGCCACGTATTACTGTGCAAGAGGGGGTTATACTATGGACTACTGGGGCCAGGGA >pVxK7b-037-hum07-HC(SEQ ID NO: 89) CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTGGGGCTACAGTGAAAATCTCCTGCAAGGTTTCTGGCTACACCTTCACCAACTACTATGTATTCTGGGTGCGACAGGCTCGTGGACAACGCCTTGAGTGGATAGGTGACATTAATCCTGTCAATGGTGATACTAACTTCAATGAGAAATTCAAGAACAGAGTCACGATTACCGCGGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAGGGGGTTATACTATGGACTACTGGGGCCAGGGA >pVxK7b-037-hum08-HC(SEQ ID NO: 90) CAGATCACCTTGAAGGAGTCTGGTCCTACGCTGGTGAAACCCACACAGACCCTCACGCTGACCTGCACCTTCTCTGGCTACACCTTCACCAACTACTATGTATTCTGGATCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGTGACATTAATCCTGTCAATGGTGATACTAACTTCAATGAGAAATTCAAGAACAGATTCACCATCTCCAGAGACAACGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGGGGGTTATACTATGGACTACTGGGGCCAGGGA >pVxK7b-037-hum09-HC(SEQ ID NO: 91) CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGACCCTGTCCCTCACCTGCACTGTCTCTGGCTACACCTTCACCAACTACTATGTATTCTGGGTGCGACAGGCTCGTGGACAACGCCTTGAGTGGATAGGTGACATTAATCCTGTCAATGGTGATACTAACTTCAATGAGAAATTCAAGAACAGACTCACCATCTCCAAGGACACCTCCAAAAACCAGGTGGTCCTTACAATGACCAACATGGACCCTGTGGACACAGCCACGTATTACTGTGCAAGAGGGGGTTATACTATGGACTACTGGGGCCAGGGA >pVxK7b-037-hum10-HC(SEQ ID NO: 92) CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGACCCTGTCCCTCACCTGCACTGTCTCTGGCTACACCTTCACCAACTACTATGTATTCTGGGTGCGACAGGCTCGTGGACAACGCCTTGAGTGGATAGGTGACATTAATCCTGTCAATGGTGATACTAACTTCAATGAGAAATTCAAGAACAGACTCACCATCTCCAAGGACACCTCCAAAAACCAGGTGGTCCTTACAATGACCAACATGGACCCTGTGGACACAGCCACGTATTACTGTGCAAGAGGGGGTTATACTATGGACTACTGGGGCCAGGGA >pVxK7b-037-hum11-HC(SEQ ID NO: 93) CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTGGGGCTACAGTGAAAATCTCCTGCAAGGTTTCTGGCTACACCTTCACCAACTACTATGTATTCTGGGTGCGACAGGCTCGTGGACAACGCCTTGAGTGGATAGGTGACATTAATCCTGTCAATGGTGATACTAACTTCAATGAGAAATTCAAGAACAGAGTCACGATTACCGCGGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAGGGGGTTATACTATGGACTACTGGGGCCAGGGA >pVxK7b-037-hum12-HC(SEQ ID NO: 94) CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTGGGGCTACAGTGAAAATCTCCTGCAAGGTTTCTGGCTACACCTTCACCAACTACTATGTATTCTGGGTGCGACAGGCTCGTGGACAACGCCTTGAGTGGATAGGTGACATTAATCCTGTCAATGGTGATACTAACTTCAATGAGAAATTCAAGAACAGAGTCACGATTACCGCGGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAGGGGGTTATACTATGGACTACTGGGGCCAGGGA >pVxK7b-037-hum13-HC(SEQ ID NO: 95) GAAGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAGGATCTCCTGTAAGGGTTCTGGCTACACCTTCACCAACTACTATGTATTCTGGATCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGTGACATTAATCCTGTCAATGGTGATACTAACTTCAATGAGAAATTCAAGAACAGAGTCACGATTACCGCGGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAGGGGGTTATACTATGGACTACTGGGGCCAGGGA >pVxK7b-037-hum14-HC(SEQ ID NO: 96) CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGACCCTGTCCCTCACCTGCACTGTCTCTGGCTACACCTTCACCAACTACTATGTATTCTGGGTGCGACAGGCTCGTGGACAACGCCTTGAGTGGATAGGTGACATTAATCCTGTCAATGGTGATACTAACTTCAATGAGAAATTCAAGAACAGAGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTACCTGCAGTGGAGCAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGAGGGGGTTATACTATGGACTACTGGGGCCAGGGA >pVxK7b-037-hum15-HC(SEQ ID NO: 97) CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGACCCTGTCCCTCACCTGCACTGTCTCTGGCTACACCTTCACCAACTACTATGTATTCTGGGTGCGACAGGCTCGTGGACAACGCCTTGAGTGGATAGGTGACATTAATCCTGTCAATGGTGATACTAACTTCAATGAGAAATTCAAGAACAGACTCACCATCTCCAAGGACACCTCCAAAAACCAGGTGGTCCTTACAATGACCAACATGGACCCTGTGGACACAGCCACGTATTACTGTGCAAGAGGGGGTTATACTATGGACTACTGGGGCCAGGGA >pVxK7b-037-hum16-HC(SEQ ID NO: 98) CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGACCCTGTCCCTCACCTGCACTGTCTCTGGCTACACCTTCACCAACTACTATGTATTCTGGGTGCGACAGGCTCGTGGACAACGCCTTGAGTGGATAGGTGACATTAATCCTGTCAATGGTGATACTAACTTCAATGAGAAATTCAAGAACAGAGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTACCTGCAGTGGAGCAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGAGGGGGTTATACTATGGACTACTGGGGCCAGGGA >pVxK7b-037-hum17-HC(SEQ ID NO: 99) GAGGTCCAGCTGGTACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCTACAGTGAAAATCTCCTGCAAGGTTTCTGGCTACACCTTCACCAACTACTATGTATTCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGTGACATTAATCCTGTCAATGGTGATACTAACTTCAATGAGAAATTCAAGAACAGAGTCACGATTACCGCGGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAGGGGGTTATACTATGGACTACTGGGGCCAGGGA >pVxK7b-037-hum18-HC(SEQ ID NO: 100) GAAGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAGGATCTCCTGTAAGGGTTCTGGCTACACCTTCACCAACTACTATGTATTCTGGATCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGTGACATTAATCCTGTCAATGGTGATACTAACTTCAATGAGAAATTCAAGAACAGAGTCACGATTACCGCGGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAGGGGGTTATACTATGGACTACTGGGGCCAGGGA >pVxK7b-037-hum19-HC(SEQ ID NO: 101) GAAGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAGGATCTCCTGTAAGGGTTCTGGCTACACCTTCACCAACTACTATGTATTCTGGATCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGTGACATTAATCCTGTCAATGGTGATACTAACTTCAATGAGAAATTCAAGAACAGAGTCACGATTACCGCGGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAGGGGGTTATACTATGGACTACTGGGGCCAGGGA >pVxK7b-037-hum20-HC(SEQ ID NO: 102) CAGATCACCTTGAAGGAGTCTGGTCCTACGCTGGTGAAACCCACACAGACCCTCACGCTGACCTGCACCTTCTCTGGCTACACCTTCACCAACTACTATGTATTCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGTGACATTAATCCTGTCAATGGTGATACTAACTTCAATGAGAAATTCAAGAACAGAGTCACGATTACCGCGGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAGGGGGTTATACTATGGACTACTGGGGCCAGGGA >pVxK7b-037-hum21-HC(SEQ ID NO: 103) CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGACCCTGTCCCTCACCTGCACTGTCTCTGGCTACACCTTCACCAACTACTATGTATTCTGGGTGCGACAGGCTCGTGGACAACGCCTTGAGTGGATAGGTGACATTAATCCTGTCAATGGTGATACTAACTTCAATGAGAAATTCAAGAACAGACTCACCATCTCCAAGGACACCTCCAAAAACCAGGTGGTCCTTACAATGACCAACATGGACCCTGTGGACACAGCCACGTATTACTGTGCAAGAGGGGGTTATACTATGGACTACTGGGGCCAGGGA >pVxK7b-037-hum22-HC(SEQ ID NO: 104) CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGACCCTGTCCCTCACCTGCACTGTCTCTGGCTACACCTTCACCAACTACTATGTATTCTGGGTGCGACAGGCTCGTGGACAACGCCTTGAGTGGATAGGTGACATTAATCCTGTCAATGGTGATACTAACTTCAATGAGAAATTCAAGAACAGACTCACCATCTCCAAGGACACCTCCAAAAACCAGGTGGTCCTTACAATGACCAACATGGACCCTGTGGACACAGCCACGTATTACTGTGCAAGAGGGGGTTATACTATGGACTACTGGGGCCAGGGA >pVxK7b-037-hum23-HC(SEQ ID NO: 105) CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGACCCTGTCCCTCACCTGCACTGTCTCTGGCTACACCTTCACCAACTACTATGTATTCTGGGTGCGACAGGCTCGTGGACAACGCCTTGAGTGGATAGGTGACATTAATCCTGTCAATGGTGATACTAACTTCAATGAGAAATTCAAGAACAGACTCACCATCTCCAAGGACACCTCCAAAAACCAGGTGGTCCTTACAATGACCAACATGGACCCTGTGGACACAGCCACGTATTACTGTGCAAGAGGGGGTTATACTATGGACTACTGGGGCCAGGGA >pVxK7b-037-hum24-HC(SEQ ID NO: 106) CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTGGGGCTACAGTGAAAATCTCCTGCAAGGTTTCTGGCTACACCTTCACCAACTACTATGTATTCTGGGTGCGACAGGCTCGTGGACAACGCCTTGAGTGGATAGGTGACATTAATCCTGTCAATGGTGATACTAACTTCAATGAGAAATTCAAGAACAGAGTCACGATTACCGCGGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAGGGGGTTATACTATGGACTACTGGGGCCAGGGAComplete Light Chain Amino Acid Sequences>VxP037-01-LC-Pro represents the full length light chain variabledomain+constant domain amino acid sequence. The underlined amino acidsequence=framework 4+the constant domain. All the humanized light chainsequences contain the same constant domain as VxP037-01-LC-Pro. However,this is not shown in the remaining humanized light chain amino acidsequences.

>VxP037-01-LC-Pro (SEQ ID NO: 107)DVVMTQTPLSLSVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSYRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQNTHVPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECComplete Light Chain Nucleic Acid Sequences

The underlined nucleic acid sequence encodes the underlined proteinsequence in >VxP037-01-LC-Pro, above.

>VxP037-01-LC-DNA (SEQ ID NO: 108)GATGTTGTTATGACCCAAACTCCACTCTCCCTGTCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCCTTGTACACAGTAATGGAAACACCTATTTACATTGGTACCTGCAGAAGCCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCTACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTTCTGCTCTCAAAATACACATGTTCCTCGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTGAComplete Heavy Chain Amino Acid Sequences

>VxP037-01-HC-Pro represents the full length heavy chain variabledomain+constant domain amino acid sequence. The underlined amino acidsequence=framework 4+the constant domain. All the humanized heavy chainsequences contain the same constant domain as >VxP037-01-HC-Pro.However, this is not shown in the remaining humanized heavy chain aminoacid sequences.

>VxP037-01-HC-Pro (SEQ ID NO: 109)EVQLQQFGAELVKPGASMKLSCKASGYTFTNYYVFWVKQRPGQGLEWIGDINPVNGDTNFNEKFKNKATLTVDKSSTTTYLQLSSLTSEDSAVYYCTRGGYTMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGKComplete Heavy Chain Nucleic Acid Sequences

The underlined nucleic acid sequence encodes the underlined proteinsequence in >VxP037-01-HC-Pro, above.

>VxP037-01-HC-DNA (SEQ ID NO: 110)GAGGTCCAGCTGCAGCAGTTTGGGGCTGAACTGGTGAAGCCTGGGGCTTCAATGAAGTTGTCCTGCAAGGCTTCTGGCTACACCTTCACCAACTACTATGTATTCTGGGTGAAACAGAGGCCTGGACAAGGCCTTGAGTGGATTGGAGACATTAATCCTGTCAATGGTGATACTAACTTCAATGAGAAATTCAAGAACAAGGCCACACTGACTGTAGACAAGTCCTCCACCACAACATACTTGCAACTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTACAAGAGGGGGTTATACTATGGACTACTGGGGCCAGGGAACGCTGGTCACCGTCAGCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCAGCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA

What is claimed is:
 1. A humanized monoclonal antibody, or antigenbinding fragment thereof, that specifically binds human, rat, mouse, andpig CD47, and wherein said humanized monoclonal antibody, orantigen-binding fragment thereof, comprises three light chaincomplementarity-determining regions (LCDRs 1-3) and three heavy chaincomplementarity-determining regions (HCDRs 1-3), wherein: LCDR1comprises the amino acid sequence RSSQSLVHSNGNTYLH (SEQ ID NO:1), LCDR2comprises the amino acid sequence KVSYRFS (SEQ ID NO:2); and LCDR3comprises the amino acid sequence SQNTHVPRT (SEQ ID NO:3); HCDR1comprises the amino acid sequence GYTFTNYYVF (SEQ ID NO:4); HCDR2comprises the amino acid sequence DINPVNGDTNFNEKFKN (SEQ ID NO:5); andHCDR 3 comprises the amino acid sequence GGYTMDY (SEQ ID NO:6).
 2. Thehumanized monoclonal antibody, or antigen binding fragment thereof, ofclaim 1, which promotes phagocytosis and/or killing of cells ofsusceptible cancer.
 3. The humanized monoclonal antibody, or antigenbinding fragment thereof, of claim 2, wherein said susceptible cancer isselected from the group consisting of a leukemia, a lymphoma, ovariancancer, breast cancer, endometrial cancer, colon cancer, rectal cancer,bladder cancer, lung cancer, bronchial cancer, bone cancer, prostatecancer, pancreatic cancer, liver and bile duct cancer, esophagealcancer, renal cancer, thyroid cancer, head and neck cancer, testicularcancer, glioblastoma, astrocytoma, melanoma, and leiomyosarcoma.
 4. Thehumanized monoclonal antibody, or antigen binding fragment thereof, ofclaim 3, wherein said leukemia is selected from the group consisting ofacute lymphocytic (lymphoblastic) leukemia, acute myeloid leukemia,chronic lymphocytic leukemia, multiple myeloma, and chronic myeloidleukemia.